Biochemistry for Medical Laboratory Science
Chapter 1: Introduction
Biochemistry


Biological Chemistry
Study of chemistry of the living organism. Includes
biomolecules, and biochemical reactions.
Four Biomolecules of the body
1. Proteins
2. Carbohydrates
3. ZxzLipids
4. Nucleic Acids
1
Base on the illustration there is a part of Hydrophilic
Hydrophilic are opposites of hydrophobic, they are water loving
molecules they don’t repel water.

Because of the presence of hydrophilic and hydrophobic
molecules on Palmitic Acid we can consider this molecule
as an Amphipathic molecule which hydrophilic and
hydrophobic are both present.
Functional Groups
are group of atoms that renders the chemical properties of
an organic molecules and biomolecules.
Biomolecules-can be found in Plant and Animal cells
Cytological composition
1. 50-95 % water
2. 1% ions- magnesium, potassium, calcium ions
3. Other organic molecules
Organic Molecules


Are Carbon based molecules
Covalently bond to itself or other elements ex. H, O, N, S,
and P
Simplest organic
molecule
CH4 (methane)
Hydrocarbons




One of the most organic molecules
Are derived from Hydrogen and Carbons
If the hydrocarbons chains are longer the more it becomes
Non-polar and insoluble in water whether there is the
presence of OH.
Note that hydrocarbons are useless inside the body, the
only thing that important in this compound is the
derivative Hydrogen and Carbon.
Hydrophobic and Hydrophilic
Hydrophobic are molecules that repels water that usually
are nonpolar molecules
Example: Lipids/ fatty acids a monocarboxylic acid can be a
component of fat/oil. A good example of this is a Palmitic acid
Note: Alcohol- OH group- Hydroxyl group
The presence of this group makes the biomolecules
abundant (can form hydrogen bonding) and polar.
Aldehyde- usually have smell (not that really good)
Acids- fatty acids are example of acids in lipids; some certain kind of
acids are found in acidic amino acids- bears the carboxyl group
(makes a weak acid)
Note: if H+ is easily remove it has a higher acidity; not all hydrogen
are acidic because it only becomes acidic when it is beside an
electronegative element.
Amines- a weak basic; had a positive charge when it accepts proton;
NH2 in amino acids.
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
Thiol- contains Sulfur; important in the proteins which it forms the
disulfide bond.
Esters- makes up fat and acid.

Double bond is important in fatty acids; reactive site or serve
as like a dipole bond in fatty acids.
1. Saturated- single bond
2. Unsaturated- double bond


Chapter 2. Water: The Solvent for Biochemical Reactions

Polarity of Water



Water is not a linear molecule.
Water molecule is bent thus, asymmetric distribution of
electron density occurs.
Oxygen of water has a high electronegativity which pulls
hydrogen electrons closer and creates a partial positive
charge.
2
When we add a non-polar substance in water, water forms
a cage around the non-polar substances. This situation is
not favorable to water, because non-polar substances
interferes with the waters ability to interact with other
water molecules.
When we add to or more non-polar substances to water,
the non-polar molecules will form together. This is
favorable because it releases the trapped water molecules
and allows them to once more form hydrogen bonds with
other water molecules.
This interaction between non-polar molecules in water are
called hydrophobic interactions and this effect is called
hydrophobic effect.
Intramolecular Bonds and Intermolecular Bonds
Strong Intermolecular Bonds



By an electron forces water molecule can strongly interact
with each other.
The partial positive charge part of hydrogen allows it to get
very close with other atoms oxygen atom.
This intermolecular bond is called a hydrogen bond.
Hydrophobic Effect



Because of the high polarity characteristic and to hydrogen
bond of water, it can easily dissolve other polar
substances.
In some situations, when we add sodium chloride in water,
water can break the ionic bonds between Na and Cl, and
form many other hydrogen bonds, this situation is
favorable.
Non-polar molecules don’t interact favorably with water.
Intramolecular Bonds – bonds that exist in any given molecules,
atoms interact with one another via these bonds.
1. Non-polar Covalent- formed between 2 two atoms by the
equal sharing of electrons. The electronegativity value of
the two atoms is equal.
2. Polar Covalent- unequal sharing of electrons that arises
due to different electronegativity values.
3. Ionic Bond- one atom with so much more electronegativity
pulls away the electron completely to its side from the
other atom.
Intermolecular Bonds – Bonds that exist between atoms of different
molecules. Considered as weaker than intermolecular bonds on a
one to one basis, there are usually many intermolecular bonds at
any given moment and this makes them a driving force in many
biochemical processes.
1. Hydrogen Bonds (dipole-dipole) - hydrogen atom is shared
by two electronegative atoms. Strongest intermolecular
bond; the group that has the H- atom is called the H-bond
donor, while the other group that accepts is the H-bond
acceptor.
2. London-Dispersion Forces (van der Waals) - the electron
density around atoms is not static but rather fluctuates
with the time. The asymmetric distribution of one
molecule can cause the electron density of a nearby
molecules to change accordingly. The two molecules can
then bond through the instantaneous dipole moments.
Acids and Bases


Determines what the pH of a solution is.
pH is a factor that can influence the many different types
of biological processes that take place inside our body.
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science

pH can determine the final structure of a biological
molecules.
Acid and Base reaction




A hydrogen atom is exchanged between molecules.
One covalent bond is broken and one is formed.
Acid molecule donates H+ ion and a bond are broken
Base molecule accepts H+ ion because it has a lone pair of
electrons and it forms covalent bond.
𝑯𝑨 ↔ 𝑯+ + 𝑨−
Acid dissociation
𝑯+ + 𝑯𝟐𝑶 ↔ 𝑯𝟐𝑶+𝑯
Base hydrogen in water exist
as hydronium molecules

The concentration of H ions is measured in terms of pH.
𝒑𝑯 = −𝒍𝒐𝒈[𝑯+]

A pH of 7.0 means that [H+] =1.0x10-7
𝟕. 𝟎 = −𝒍𝒐𝒈[𝑯+] → 𝟕. 𝟎 = 𝒍𝒐𝒈[𝑯+] → 𝟏𝟎−𝟕 = [𝑯+]

We can also use the pH to describe the concentration of
OH- in solution
𝑯𝟐𝑶 ↔ 𝑯+ + 𝑶𝑯−
[𝑯+][𝑶𝑯−]
𝑲=
[𝑯𝟐𝑶]

At room temperature, K=1.8x10-16 and the concentration
of H2O in pure water is always equal to a constant value of
55.5
[𝑯+][𝑶𝑯−]
𝟏. 𝟖𝒙𝟏𝟎−𝟏𝟔 =
= 𝟏. 𝟎𝒙𝟏𝟎−𝟏𝟒 = [𝑯+][𝑶𝑯−]
𝟓𝟓. 𝟓

Therefore, at a pH=7, the concentration of hydroxide is
also 1.0x10-7
𝟏. 𝟎𝒙𝟏𝟎−𝟏𝟒 = (𝟏. 𝟎𝒙𝟏𝟎−𝟕)[𝑶𝑯−] → [𝑶𝑯−] = 𝟏. 𝟎𝒙𝟏𝟎−𝟕
Amino Acid Structure and Properties

With the exception of glycine, all protein-derived amino
acids have at least one stereo center (the a-carbon) and
are chiral (stereoisomers)
o the vast majority of a-amino acids have the Lconfiguration at the a-carbon (Proline is usually D)

Side-chain carbons in other amino acids designated with
Greek symbols, starting at a carbon (…etc)

Amino acids can be referred to by three-letter or oneletter codes.
Individual Amino Acids
Group A: Nonpolar Side Chains
1. Alanine – Ala – A
2. Valine – Val – V
3. Leucine – Leu – L
4. Isoleucine – Ile – I
5. Proline – Pro – P
6. Phenylalanine – Phe – F
7. Tryptophan – Trp – W
8. Methionine – Met – M
Chapter 3. Amino Acids and Peptides
Amino Acids Exist in a 3-D World
Amino acid: a compound that contains both an amino group and a
carboxyl group

c-Amino acid has an amino group attached to the carbon
adjacent to the carboxyl group

-carbon also bound to side chain group, R

R gives identity to amino acid

Two stereoisomers of amino acids are designated L- or D-.
Based on similarity to glyceraldehyde
Important Structural Features:
1.
2.
3.
4.
5.
All 20 are a-amino acids
For 19 of the 20, the a-amino group is primary; for proline,
it is secondary
With the exception of glycine, the a-carbon of each is a
stereocenter
Isoleucine and threonine contain a second stereocenter
3, and 1-letter codes (ex. Glycine – Gly – G)
Amino acids
Ala, Val, Leu, Ile, Pro
Pro
Phe
Trp
Met
Features
contain aliphatic hydrocarbon group
Pro has cyclic structure
hydrocarbon aromatic ring
Indole ring side chain, aromatic
Sulfur atom in side chain
Group B: Neutral Polar Side Chains
1. Serine – Ser – S
2. Threonine – Thr – T
3. Tyrosine – Tyr – Y
4. Cysteine – Cys – C
5. Glutamine – Gln – Q
6. Asparagine – Asn – N
Amino acids
Features
Ser, Thr
Side chain is polar hydroxyl group
Tyr
hydroxyl group bonded to aromatic
hydrocarbon group
Cys
Side chain contains thiol group (-SH)
Gln, Asn
contain amide bonds in side chain
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
3
Biochemistry for Medical Laboratory Science
4
Group C: Acidic Side Chains
1. Glutamic Acid – Glu – E
2. Aspartic Acids – Asp – D
 Both have a carboxyl group in side chain
 Can lose a proton, forming a carboxylate ion
 These amino acids are negatively charged at neutral pH
Group D: Basic Side Chains
1. Histidine – His – H
2. Lysine – Lys – K
3. Arginine – Arg – R
 Side chains are positively charged at pH 7
Amino acids
Features
Arg
side chain is a guanidino group
His
side chain is an imidazole group
side chain NH3 group is attached to an aliphatic
Lys
hydrocarbon chain
Ionization of Amino Acids
Remember, amino acids without charged groups on side
chain exist in neutral solution as zwitterions with no net charge
Acidity: -COOH Groups
The average pKa of a α-carboxyl group is 2.19, which makes
them considerably stronger acids than acetic acid (pKa 4.76)

the greater acidity of the amino acid carboxyl group is due
to the electron-withdrawing inductive effect of the -NH 3+
group
Basicity
α-NH3+ groups: The average value of pKafor an a-NH 3+ group
is 9.47, compared with a value of 10.76 for a 2° alkylammonium ion
Guanidine Group

The side chain of arginine is a considerably stronger base
than an aliphatic amine
o basicity of the guanido group is attributed to the large
resonance stabilization of the protonated form
relative to the neutral form
Imidazole Group

The side chain imidazole group of histidine is a heterocyclic
aromatic amine
Titration of Amino Acids
When an amino acid is titrated, the titration curve
represents the reaction of each functional group with the hydroxide
ion
Uncommon Amino Acids
Each derived from a common amino acid by a modification:

hydroxylysine and hydroxyproline are found only in a
few connective tissues such as collagen

thyroxine is found only in the thyroid gland
Titration of Histidine with NaOH
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
5
Chapter 4. The Three-Dimensional Structure of Protein
Primary Structure
these are the sequence made up of specific amino acids.

Ionization of Amino Acids

In amino acid, carboxyl group (-) and amino group (+) are
charged at neutral pH.

In free amino acids -carboxyl, and a-amino groups have
titratable protons. Some side chains do as well


Isoelectric pH
Isoelectric pH, pI: the pH at which the majority of
molecules of a compound in solution have no net charge

The linear polymers of amino acids contain polarity. By contains
polarity. By concentration, the beginning of any polypeptide
chain is at the α-amino group and the end is at the end is at the
α-carboxyl group.
Each amino acid is called a residue. The polypeptide chain
consists of separating units that makes up the backbone. The
variable portions of the polypeptide are the side chains.
The polypeptide chain has the ability to form more hydrogen
bonds.
a. Hydrogen-bond donor: N-H group
b. Hydrogen-bond acceptor: C=O group
the pI for glycine, for example, falls midway between the pKa
values for the carboxyl and amino groups
The Peptide Bond


Individual amino acids can be linked by forming covalent
bonds.
Peptide bond: the special name given to the amide bond
between the a-carboxyl group of one amino acid and the
a-amino group of another amino acid
Direction of Peptide Chain

Peptide bonds are resonance-stabilized, which means they have
double-bond character.

The double bond nature of the peptide bond:
1. Makes the peptide bond planar.
2. Prevents any rotation about the peptide bond.
Trans and Cis Configuration
Majority of the cases, trans peptide bonds are energetically
more favorable than cis because these is no bumping of atoms.
Geometry of Peptide Bond



the four atoms of a peptide bond and the two alpha carbons
joined to it lie in a plane with bond angles of 120°about C
and N
to account for this geometry, a peptide bond is most
accurately represented as a hybrid of two contributing
structures (resonance structures)
the hybrid has considerable C-N double bond character and
rotation about the peptide bond is restricted
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science

Secondary Structure
Once the primary structure of the polypeptide is formed, is
begins to twist into regular patterns that make up the secondary
structure. These include the α-helix, the β-pleated sheet, the B-turn,
and Ω loop. These twists are found as a result of the regular pattern
of hydrogen bonds between NH and C=O groups on the polypeptide
chain.
6
In this arrangement, the NH and C=O groups of an amino
acid on one strand form H bonds with C=O and N-H groups
of the opposing amino acid on the other strand.
Parallel Beta Sheet

In the parallel beta sheet, the adjacent strands run in the
same direction.

An amino acid on one strand connects to two amino acids on
the opposing end via hydrogen bond.
Alpha Helix




The alpha-helix is a rod-like
structure that contains the
backbone on the inner portion of
the helix and the side chains on the
outer portion.
Each amino acid uses its N-H group
to form a hydrogen bond with the
C=O of the amino acid that is four
units ahead of it.
The screw sense of the α-helix
describes the direction in which the
helix rotates with respect to its axis.
A right-handed helix rotates
clockwise while a left-handed helix
rotates counter clockwise.
The right-handed α-helix
predominates because there is less
steric hindrance between the side
chains.
Beta Turns

The compact nature of proteins is in part due to the
polypeptides ability to make sudden turns in their chain.

These turns, called B turns or reverse turns, are stabilized by
hydrogen bonding. They allow polypeptide to make abrupt
turns and are usually found on the surface of the protein.
Beta Pleated Sheets
Tertiary Structure
The tertiary structure refers to the spatial arrangement of
amino acids that are found far away from on another along the
polypeptide chain
Antiparallel Beta Sheet

In the antiparallel beta sheet, two linear polymers of amino
acids run in opposite directions.
Hydrophobic Interactions

Most proteins exist in an aqueous solution. We know that when
non-polar molecules are placed into water, they will aggregate
together because this will create a thermodynamically more
stable system.
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science

What does that tell us about the way in which the polypeptide
will fold?
1. Those amino acids with hydrophobic side chains (i.e.
valine, leucine, etc.) will tend to be found inside the
protein.
2. Those amino acids that contain hydrophilic side chain (i.e.
lysine, aspartate) will tend to be found on the outside of
the protein,
7
Fibrous proteins

These large proteins form long fibers and play a structural role.
Keratin and collagen are two examples of these proteins.
Keratin

van der Waal’s Interaction
The non-polar amino acids of the protein core interact
with one another via their instantaneous dipole moments. Although
these van der Waals forces are relatively weak on an individual base,
the aggregate effect of the many non-polar amino acids creates a
substantial binding effect.
α-Keratin is the major component of hair and it consists of two
polypeptide subunits. These subunits consist of right-handed αhelices that intertwine to form a left-handed supercoil called
the α-coiled coil.
These two subunits are held together by:
1. van der Waals forces
2. Ionic bonds
3. Disulfide bonds
Disulfide Bonds
Globular Proteins – these proteins have a wide range of function
and are relatively spherical in shape. Some examples include
hemoglobin, insulin, DNA polymerase, etc.
Hemoglobin

In some proteins, usually the ones destined to be extracellular,
the polypeptide chains can be cross-linked via disulfide bonds
between cysteine residues. This cross-linked units are called
cystines.
Hydrogen bonds – the polar and hydrophilic side chains on the
surface interact with the water molecules via hydrogen bonds.
Ionic Interactions – two oppositely charged side chain can interact
via ionic bonds. For instance, Lysine can form an ionic bond with
aspartate

Quaternary Structure





Refers to the ways in which in these polypeptides interact with
one another.
A dimer is the simplest case of quaternary structure. In a dimer,
there are two polypeptide that constitute the protein.
Generally, each individual polypeptide is called subunit.
These subunits are usually held together by non-covalent bonds
but can also be held by covalent bonds such as disulfide bridge
There are two major categories of proteins with quaternary
structure globular and fibrous proteins.

Hemoglobin is a tetramer that consists of four individual
subunits.
Each subunit is equipped with a heme group that is capable of
binding an oxygen molecule.
Heme group consist of an organic component called
protoporphyrin as well as an inorganic component that consist
of an iron atom.
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
8
Carbonic Anhydrase catalyzes the conversion of non-polar carbon
dioxide into carbonic acid, which dissociates into the polar
bicarbonate. These enzymes speed up the reaction by a factor of
one million. In the absence of this enzyme, we would not be able to
dissolves CO3 in our blood.

Enzymes typically help transform one energy form into a
more usable form.

Plants use a variety of enzymes to capture the energy stored
in light and transform it into a more usable form. That is, into
the energy store din chemical bonds (i.e. Glucose). This
process is called photosynthesis.
𝑪𝑶𝟐 + 𝑯𝟐𝑶 + 𝒍𝒊𝒈𝒉𝒕 ↔ 𝑪𝟔𝑯𝟏𝟐𝑶𝟔 + 𝑶𝟐





The iron atom lies at the center of the protoporphyrin and is
bound to four nitrogen atoms. At this stage, iron is in its ferrous
state and has an oxidation state of +2. This means it can form
two more bonds.
On one side of the protoporphyrin plane the iron bound to the
histidine residue of the protein. This is known as the proximal
histidine
In deoxyhemoglobin the iron atom remains unbound to oxygen.
This state that Fe atom is too large to fit into the center of the
protoporphyrin ring and so the iron remains below the plane of
the protoporphyrin.
The binding of the oxygen atom to the iron pulls away electrons
from the iron, making it smaller. This allows it to fit into the
center of the protoporphyrin plane.
The actual structure of the iron-oxygen complex is resonance
stabilized, as shown above. Notice that the superoxide form has
a negative charge. This region is stabilized by another histidine
residue called the distal histidine.

Enzymes
In animals, the energy stored in the chemical bonds of
glucose, is transformed in the energy stored in the proton
gradient across the mitochondrial and this energy us then
transformed into the chemical bonds of adenosine -51triphosphate molecules.
Enzymes typically do not act alone; they require helper molecules.

Cofactor are helper molecules that are needed for enzymes
to function properly.
𝑨𝒑𝒐𝒆𝒏𝒛𝒚𝒎𝒆 + 𝑪𝒐𝒇𝒂𝒄𝒕𝒐𝒓 ↔ 𝑯𝒂𝒍𝒐𝒆𝒏𝒛𝒚𝒎𝒆

Cofactors come in two categories: metal ions and organic
molecules called coenzymes. Coenzymes can bind to
enzymes weakly or strongly. Tightly bound coenzymes are
called prosthetic groups.
Enzymes are highly specific.

Enzymes bind to specific reactants (also called substrates)
and catalyze a single reaction or a set of related reaction.
Enzymes are highly efficient and limit the number of
unwanted products.

Trypsin, a digestive enzyme, binds to polypeptide and carries
out two closely related reactions. It catalyzes the cleavage of
peptide bonds on the carboxyl side of lysine and arginine
amino acids.
Nearly all enzymes are proteins. Although the majority of enzymes
are polypeptides, some RNA molecules can also act as catalyst.
Noe: Enzymes are not used up and remain unchanged at the end of
a reaction.
Gibbs Free-Energy
Enzymes catalyze reactions and reactions are described
and studied by using thermodynamics. We can use
thermodynamically quantities such as enthalpy, entropy, Gibbs free
energy to study and understand the activity of enzymes.
Gibbs free energy
𝑹𝒆𝒂𝒄𝒕𝒂𝒏𝒕𝒔 ↔ 𝑷𝒓𝒐𝒅𝒖𝒄𝒕𝒔
Behavior of Proteins: Enzymes
Enzymes

Biological molecule that speeds up biological reaction.

Catalyzes reactions that take place in our cells.

Without these cellular processes would halt

They increase the rate at which reactions take place.
𝑪𝑶𝟐 + 𝑯𝟐𝑶 ↔ 𝑯𝟐𝑪𝑶𝟑 ↔ 𝑯+ + 𝑯𝑪𝑶−𝟑
Carbonic Anhydrase
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science






In chemical reactions that have not yet activated equilibrium,
the ∆𝐺 can either be positive or negative.
Gibbs free energy can be used to determine the spontaneity of
the reaction.
A chemical reaction is said to be exergonic and spontaneous if
the ∆𝐺 is negative. Combustion reactions are examples of
exergonic reactions.
On the other hand, chemical reaction is said to be endergonic
and spontaneous if the ∆𝐺 is positive. ATP synthesis is an
endergonic reaction.
Gibbs free energy depends on the energy of products and
reactants and is independent of the pathway that was taken to
convert the reactants to products.
If the ∆𝐺=0, the reaction has reached equilibrium and is said to
be neither spontaneous nor non-spontaneous. In such a case of
the forward reaction is equal to the rate of the reverse
reaction.
Active sites, Lock and Key Model, and Induced Fit Model
1.
2.
3.
4.
5.
6.
Active site is a specific region on enzyme that binds to the
substrate.
Active sites stabilize the transition state.
Active Sties establish Microenvironments
Active site makes up only a small component of the overall
enzyme.
Active sites typically bind substrates reversibly via noncovalent forces.
Active site has structures complementary to their
corresponding substrates.
Induced Fit Model – In the induced fit model, the shape of the
enzyme’s active site is not exactly complementary. However upon
binding of substrate to active site, the binding causes the active site
to become complementary to the substrate.
Michaelis-Menten Equation

The curve shows when we increase the concentration of
substrate, the velocity approaches the Vmax asymptotically.
[𝑺]
𝑽𝒐 = 𝑽𝒎𝒂𝒙
𝑲𝒏 + [𝑺]

The equation can be used to describe the enzyme activity of the
beginning reaction
Also describe the enzyme activity towards the end of the
reaction

Lock-and-Key Model – in this model the substrate first precisely and
perfectly into their exact complementary shapes.
9
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
Irreversible, and Reversible Inhibition

Irreversible Inhibition

Some inhibitors will bind to enzymes very tightly by covalent
and non-covalent bond.

When bound, they will no dissociate easily from the enzyme.


Reversible Inhibition – Exact opposite of irreversible inhibition; can
be easily dissociate from the enzyme.
Competitive inhibition

Inhibitor molecule resembles the substrate and has the
ability to fit to the active site of the enzyme.

It occupies the active site therefore prevents substrate from
binding to enzyme.
Uncompetitive inhibition

Some situation when the binding of substrate occurs,
changes of conformation of the enzyme may occur resulting
to an allosteric site that was not in there previously.

Some inhibitors can bind to the new site.
10
Only special molecules can bind to these sites and alter the
activity of the enzyme.
It shows cooperativity in proteins and enzymes.
Aspartate transcarbamoylase is an example of a allosteric
enzyme.
Reversible Covalent Modification

Some enzyme’s activity can be altered through covalently
modifying the structure of the enzyme.

Example of this is the attachment of phosphoryl group onto an
enzyme.

Protein kinases are usually used to catalyze the transfer of a
phosphoryl group from ATP onto the enzyme.

To reverse the transfer protein phosphatases can be used to do
that.
Proteolytic Cleavage

Some enzymes are produced in inactive state/form. They are
called zymogens or proenzymes.

In activating zymogens, they are typically cleaved irreversibly by
proteases at specific sites on the polypeptide.

When zymogens activated, they can eventually be inactive by
the binding of some irreversible inhibitor.

Typically, digestive enzymes and the blood cascade enzymes
use this form of regulation.
Enzyme Concentration – by regulation the transcription of specific
genes, we can control how much enzyme is produced. This can turn
regulate the overall level of activity due to some enzyme.
Isoenzymes (isozyme)

Are enzymes that differ in their amino acid sequence and
structure but which catalyze the same reaction. These enzymes
allow for the fine-tuning of many metabolic processes.

Isoenzymes usually exhibit different enzyme kinetics and are
controlled by different regulatory molecules.

Lactate dehydrogenase (LDH) is an example of an enzyme that
forms.
Non-Competitive Inhibition

Enzymes has permanent allosteric site which inhibitors bind

These inhibitors do not compete for the existing active site
instead, they can bind to the existing allosteric site.
Enzyme Regulation
Allosteric control

Some enzymes contain sites (other than active sites) that also
used regularly and purposely. These are called allosteric
enzymes.
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
LIPIDS

marginally soluble (at best) in water but readily soluble in
organic solvents

a mixed bag of compounds that share some properties
based on structural similarities, mainly a preponderance of
nonpolar groups
Classified according to their chemical nature, lipids fall into two main
groups:
1. Open-chain Compounds – with polar head groups and long
nonpolar tails; includes fatty acids, triacylglycerols,
sphingolipids, phosphoacylglycerols, and glycolipids
2. Fused-ring Compounds – the steroids; an important
representative of this group is cholesterol
The Chemical Nature of the Lipid Types
Fatty Acids – has a carboxyl group at the polar end and a hydrocarbon
chain at the nonpolar tail

They are amphipathic compounds – the carboxyl group is
hydrophilic and the hydrocarbon tail is hydrophobic

A fatty acid that occurs in a living system normally contains
an even number of carbon atoms, and the hydrocarbon
chain is usually unbranched

Saturated – there are only single bonds

Unsaturated – there are carbon-carbon double bonds in the
chain; the stereochemistry at the double bond is usually cis
rather than trans
o cis – puts a kink in the long-chain hydrocarbon tail
o trans – the shape is like that of a saturated fatty
acid in its fully extended conformation

Fatty acids are rarely found free in nature, but they form
parts of many commonly occurring lipids
Typical naturally occurring unsaturated fatty acids
Acid
Number
of C
Atoms
Palmitoleic
16
CH3(CH2)5CH=CH(CH2)7CO2H
-0.5
Oleic
18
CH3(CH2)7CH=CH(CH2)7CO2H
16
Linoleic
18
CH3(CH2)4CH=CH(CH2)CH=CH(CH2)7CO2H
-5
Linolenic
18
CH3(CH2CH=CH)3(CH2)7CO2H
-11
Arachidonic
20
CH3(CH2)4CH=CH(CH2)4(CH2)2CO2H
-50
Formula
Melting
Point
(°C)
Triacylglycerols – resulting compound when all three of the alcohol
groups form ester linkages with fatty acids

Glycerol is a simple compound that contains three hydroxyl
groups

Triglyceride – older name for triacylglycerol

The three ester groups are the polar part of the molecule,
whereas the tails of the fatty acids are nonpolar

Triacylglycerols do not occur as components of membranes
(as do other types of lipids), but they accumulate in adipose
tissue (primarily fat cells) and provide a means of storing
fatty acids, particularly in animals


Structures of some typical fatty acids
11
When an organism uses fatty acids, the ester linkages of
triacylglycerols are hydrolyzed by enzymes called lipases
When a base such as sodium hydroxide or potassium
hydroxide is used, the products of the reaction, which is
called saponification, are glycerol and the sodium or
potassium salts of the fatty acids
Typical naturally occurring saturated fatty acids
Acid
Number of
Carbon Atoms
Lauric
12
CH3(CH2)10CO2H
44
Myristic
14
CH3(CH2)12CO2H
58
Palmic
16
CH3(CH2)14CO2H
63
Stearic
18
CH3(CH2)16CO2H
71
Arachidic
20
CH3(CH2)18CO2H
77
Formula
Melting
Point (°C)
Hydrolysis of triacylglycerols
Phosphoacylglycerols

Phosphatidic acid – resulting compound when one of the
alcohol groups of glycerol is esterified to a phosphoric acid
molecule rather than to a carboxylic acid
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science

Phosphatidyl ester – one molecule of phosphoric acid
forms ester bonds both to glycerol and to some other
alcohol; classed as phosphoacylglycerols
12
Sphingolipids – do not contain glycerol, but they do contain the longchain amino alcohol sphingosine

Sphingolipids are found in both plants and animals; they are
particularly abundant in the nervous system

In sphingomyelins, the primary alcohol group of
sphingosine is esterified to phosphoric acid, which, in turn,
is esterified to another amino alcohol, choline
Structures of some waxes and sphingolipids



The classification of a phosphatidyl ester depends on the
nature of the second alcohol esterified to the phosphoric
acid
Some of the most important lipids in this class are
phosphatidyl ethanolamine (cephalin), phosphatidyl
serine, phosphatidyl choline (lecithin), phosphatidyl
inositol, phosphatidyl glycerol, and diphosphatidyl
glycerol (cardiolipin)
All these compounds have long, nonpolar, hydrophobic tails
and polar, highly hydrophilic head groups and thus are
markedly amphipathic
Structures of some phosphoacylglycerols
Glycolipids – the resulting compound if a carbohydrate is bound to an
alcohol group of a lipid by a glycosidic linkage

Ceramides – the parent compounds for glycolipids

Cerebroside – the glycosidic bond is formed between the
primary alcohol group of the ceramide and a sugar residue

In most cases, the sugar is glucose or galactose; for
example, a glucocerebroside is a cerebroside that contains
glucose


Cerebrosides are found in nerve and brain cells, primarily in
cell membranes
Gangliosides – examples of glycolipids with a complex
carbohydrate moiety that contains more than three sugars;
also present in large quantities in nerve tissues
Waxes and Sphingolipids
Waxes – complex mixtures of esters of long-chain carboxylic acids and
long-chain alcohols

They frequently serve as protective coatings for both plants
and animals
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
13
Steroids

Many compounds of widely differing functions are classified
as steroids because they have the same general structure:
a fused-ring system consisting of three six-membered rings
(the A, B, and C rings) and one five-membered ring (the D
ring)

Cholosterol – widespread in biological membranes,
especially in animals, but it does not occur in prokaryotic
cell membranes; has a number of important biological
functions, including its role as a precursor of other steroids
and of vitamin D3

Cholesterol plays a role in the development of
atherosclerosis, a condition in which lipid deposits block the
blood vessels and lead to heart disease
Structures of some steroids
Lipid bilayers
(A) The fused-ring structure of steroids. (B) Cholesterol. (C) Some
steroid sex hormones.
Biological Membranes

The molecular basis of the membrane’s structure lies in its
lipid and protein components

Membranes not only separate cells from the external
environment, but also play important roles in the transport
of specific substances into and out of cells

Phosphoglycerides are prime examples of amphipathic
molecules, and they are the principal lipid components of
membranes

The existence of lipid bilayers depends on hydrophobic
interactions

The most important difference between lipid bilayers and
cell membranes is that the latter contain proteins as well as
lipids
Lipid Bilayers

The polar head groups are in contact with water, and the
nonpolar tails lie in the interior of the membrane

The whole bilayer arrangement is held together by
noncovalent interactions, such as van der Waals and
hydrophobic interactions

The surface of the bilayer is polar and contains charged
groups

The nonpolar hydrocarbon interior of the bilayer consists of
the saturated and unsaturated chains of fatty acids and the
fused-ring system of cholesterol
Lipid bilayer asymmetry. The compositions of the outer and inner
layers differ; the concentration of bulky molecules is higher in the
outer layer, which has more room.
Membrane Fluidity

The arrangement of the hydrocarbon interior of the bilayer
can be ordered and rigid or disordered and fluid

In saturated fatty acids, a linear arrangement of the
hydrocarbon chains leads to close packing of the molecules
in the bilayer, and thus to rigidity

Unsaturated fatty acids have a kink in the hydrocarbon
chain that does not exist in saturated fatty acids
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science


The disordered structure caused by the presence of
unsaturated fatty acids with cis double bonds (and
therefore kinks) in their hydrocarbon chains causes greater
fluidity in the bilayer
The lipid components of a bilayer are always in motion, to a
greater extent in more fluid bilayers and to a lesser extent
in more rigid ones
Membrane Proteins
Proteins in the biological membrane
1. Peripheral proteins – on the surface of the membrane;
loosely bound to the outside of a membrane
2. Integral proteins – within the lipid bilayer; embedded in a
membrane
3.
Receptor Property – proteins bind specific biologically
important substances that trigger biochemical responses in
the cell
Membrane Transport
1. Passive Transport – a substance moves from a region of
higher concentration to one of lower concentration;
movement of the substance is in the same direction as a
concentration gradient; cell does not expend energy
a. Simple Diffusion – a molecule moves directly
through the membrane without interacting with
another molecule
b. Facilitated Diffusion – the process of moving a
molecule passively through a membrane using a
carrier protein, to which molecules bind
2.
Proteins in the membrane according to its function
1. Transport proteins – help move substances in and out of
the cell
2. Receptor proteins – important in the transfer of
extracellular signals, such as those carried by hormones or
neurotransmitters, into the cell
14
Active Transport – a substance moves from a region of
lower concentration to one of higher concentration (against
a concentration gradient); requires the cell to expend
energy

Sodium-potassium ion pump – the export of
sodium ions from a cell with simultaneous inflow
of potassium ions, both against concentration
gradients
Fluid-Mosaic Model of Membrane Structure

The most widely accepted description of biological
membranes

The model for membrane structure in which proteins and a
lipid bilayer exist side by side without covalent bonds
between the proteins and lipids

The proteins “float” in the lipid bilayer and can move along
the plane of the membrane
The Functions of Membranes
1. Transport – membranes are semipermeable barriers to the
flow of substances into and out of cells and organelles
2. Catalysis – enzymes can be bound—in some cases very
tightly—to membranes, and the enzymatic reaction takes
place on the membrane
Membrane Receptor

The first step in producing the effects of some biologically
active substances is binding the substance to a protein
receptor site on the exterior of the cell

The interaction between receptor proteins and the active
substances that bind to them has features in common with
enzyme–substrate recognition

There is a requirement for essential functional groups that
have the correct three- dimensional conformation with
respect to each other
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
Lipid-Soluble Vitamins
– are hydrophobic, which accounts for their solubility
1. Vitamin A – the lipid-soluble compound responsible for the
primary photochemical event in vision

B-carotene – an unsaturated hydrocarbon; the
precursor of vitamin A

Retinol – the alcohol form of vitamin A

Retinal – the aldehyde form of vitamin A

A derivative of vitamin A plays a crucial role in
vision when it is bound to a protein called opsin

Rhodopsin – the product of the reaction between
retinal and opsin
2.
Vitamin D – plays a major role in the regulation of calcium
and phosphorus metabolism
Vitamin D3 – cholecalciferol; formed from

cholesterol by the action of ultraviolet radiation
from the Sun; further processed in the body to
form hydroxylated derivatives, which are the
metabolically active form of this vitamin

Rickets – deficiency of vitamin D; a condition in
which the bones of growing children become soft,
resulting in skeletal deformities
4.
15
Vitamin K – an important factor in the blood-clotting
process; came from the Danish Koagulation
Prostaglandins and Leukoterines
Prostaglandins – derivatives of arachidonic acid that contain a fivemembered ring and are of pharmaceutical importance

They were first detected in seminal fluid, which is produced
by the prostate gland

Arachidonic acid – metabolic precursor of all
prostaglandins; a fatty acid that contains 20 carbon atoms
and four double bonds


Some of the functions of prostaglandins are control of blood
pressure, stimulation of smooth-muscle contraction, and induction of inflammation
Prostaglandins are known to inhibit the aggregation of
platelets
Leukotrienes – derived from arachidonic acid; found in leukocytes
(white blood cells) and have three conjugated double bonds

An important property of leukotrienes is their constriction
of smooth muscle, especially in the lungs

Leukotrienes may also have inflammatory properties and
may be involved in rheumatoid arthritis
3.
Vitamin E – a lipid-soluble antioxidant

A-tocopherol – most active form of vitamin E
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
16
LIPID METABOLISM

Lipids are involved in the generation and storage of energy

The catabolic oxidation of lipids releases large quantities of
energy, whereas the anabolic formation of lipids represents
an efficient way of storing chemical energy
Catabolism of Lipids

The oxidation of fatty acids is the chief source of energy in
the catabolism of lipids

Both triacylglycerols, which are the main storage form of
the chemical energy of lipids, and phosphoacylglycerols,
which are important components of biological membranes,
have fatty acids as part of their covalently bonded
structures

The bond between the fatty acid and the rest of the
molecule can be hydrolyzed, with the reaction catalyzed by
suitable groups of enzymes—lipases, in the case of
triacylglycerols, and phospholipases, in the case of
phosphoacylglycerols
Fatty Acid Transport into Mitochondria

Fatty acid oxidation begins with activation of the molecule

Activation in lipid metabolism involves the formation of a
thioester bond between the carboxyl group of the fatty acid
and the thiol group of coenzyme A (CoA-SH)

The activated form of the fatty acid is an acyl-CoA
The role of carnitine in the transfer of acyl groups to the
mitochondrial matrix
Oxidation of Saturated Fatty Acids

In the matrix, a repeated sequence of reactions successively
cleaves two-carbon units from the fatty acid, starting from
the carboxyl end

This process is called b-oxidation, since the oxidative
cleavage takes place at the b-carbon of the acyl group
esterified to CoA

The b-carbon of the original fatty acid becomes the carboxyl
carbon in the next stage of degradation

The whole cycle requires four reactions:
1.
2.
3.









The enzyme that catalyzes formation of the ester bond, an
acyl-CoA synthetase, requires ATP for its action
The acyl group is then transferred to CoA- SH
The esterification takes place in the cytosol, but the rest of
the reactions of fatty acid oxidation occur in the
mitochondrial matrix
The acyl-CoA can cross the outer mitochondrial membrane
but not the inner membrane
In the intermembrane space, the acyl group is transferred
to carnitine by transesterification; this reaction is catalyzed
by the enzyme carnitine acyltransferase, which is located in
the inner membrane
Acyl-carnitine, a compound that can cross the inner
mitochondrial membrane, is formed
This enzyme has a specificity for acyl groups between 14
and 18 carbons long and is often called carnitine
palmitoyltransferase (CPT-I) for this reason
The acyl-carnitine passes through the inner mem- brane via
a specific carnitine/acyl-carnitine transporter called
carnitine translocase
Once in the matrix, the acyl group is transferred from
carnitine to mitochondrial CoA-SH by another
transesterification reaction, involving a second carnitine
palmitoyltransferase (CPT-II) located on the inner face of
the membrane
4.
2.
The acyl-CoA is oxidized to an a, b unsaturated acyl-CoA (also
called a Δ-enoyl-CoA). The product has the trans arrangement
at the double bond. This reaction is catalyzed by an FADdependent acyl-CoA dehydrogenase.
The unsaturated acyl-CoA is hydrated to produce a bhydroxyacyl-CoA. This reaction is catalyzed by the enzyme
enoyl-CoA hydratase.
A second oxidation reaction is catalyzed by b-hydroxyacylCoA dehydrogenase, an NAD1-dependent enzyme. The
product is a b-ketoacyl-CoA.
The enzyme thiolase catalyzes the cleavage of the b-ketoacylCoA; a molecule of CoA is required for the reaction. The
products are acetyl-CoA and an acyl-CoA that is two carbons
shorter than the original molecule that entered the boxidation cycle. The CoA is needed in this reaction to form the
new thioester bond in the smaller acyl-CoA molecule. This
smaller molecule then undergoes another round of the boxidation cycle.
When a fatty acid with an even number of carbon atoms
undergoes successive rounds of the b-oxidation cycle, the
product is acetyl-CoA
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
The Energy Yield from the Oxidation of Fatty Acids

In aerobic processes—namely, the citric acid cycle and
oxidative phosphorylation—the energy released by the
oxidation of acetyl-CoA formed by b-oxidation of fatty acids
can also be used to produce ATP

There are two sources of ATP to keep in mind when
calculating the overall yield of ATP:
1. Reoxidation of the NADH and FADH2 produced by the
b-oxidation of the fatty acid to acetyl-CoA
2. ATP production from the processing of the acetyl-CoA
through the citric acid cycle and oxidative
phosphorylation


The process of b-oxidation gives rise to unsaturated fatty
acids in which the double bond is in the trans arrangement,
whereas the double bonds in most naturally occurring fatty
acids are in the cis arrangement
For monounsaturated fatty acids to be broken down to
acetyl-CoA, enoyl-CoA isomerase catalyzes a cis-trans
isomerization, which allows enoyl-CoA hydratase to hydrate
the trans-double bond
The Balance Sheet for Oxidation of One Molecule of Stearic Acid (an
18-carbon compound)
Reaction
1. Stearic acid S Stearyl-CoA
(activation step)
2. Stearyl-CoA S 9 Acetyl-CoA (8
cycles of b-oxidation)
3. 9 Acetyl-CoA S 18 CO2 (citric
acid cycle); GDP S GTP (9
molecules)
4. Reoxidation of NADH from boxidation cycle
5. Reoxidation of NADH from
citric acid cycle
6. Reoxidation of FADH2 from boxidation cycle
7. Reoxidation of FADH2 from
citric acid cycle
Net
NADH
Molecules
FADH2
Molecules
ATP
Molecules
18
18
22
1 27
19
In the case of oleoylCoA, three b-oxidation
cycles produce three
molecules of acetyl-CoA
and leave cis-D3dodecenoyl-CoA.
Rearrangement of
enoyl-CoA isomerase
gives the trans-D2
species, which then
proceeds normally
through the b-oxidation
pathway.
19
28
1 20
2 27
1 67.5
0
28
1 12
29
1 13.5
0
1 120
Catabolism of Unsaturated Fatty Acids and Odd-Carbon Fatty
Acids

Odd-numbered fatty acids also undergo b-oxidation

The last cycle of b-oxidation produces one molecule of
propionyl-CoA

In this pathway, propionyl-CoA is first carboxylated to
methyl malonyl-CoA in a reaction catalyzed by propionylCoA carboxylase, which then undergoes rearrangement to
form succinyl-CoA
Oxidation of Monounsaturated Fatty Acids

The conversion of a monounsaturated fatty acid to acetylCoA requires a reaction that is not encountered in the
oxidation of saturated acids, a cis–trans isomerization
17
Oxidation of Polyunsaturated Fatty Acids

When polyunsaturated fatty acids are b-oxidized, another
enzyme is needed to handle the second double bond

For fatty acids
containing
multiple double
bonds to be
catabolized, the
enoyl-CoA
isomerase is
required for the
first double bond
and 2,4-dienoylCoA reductase is
required for
subsequent
double bonds

The 2,4-dienoylCoA reductase
converts two
conjugated pi
bonds into a
single pi bond that
can be isomerized
by enoyl-CoA
isomerase, which
allows b-oxidation
to continue
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
Ketone Bodies
– substances related to acetone; produced when an excess of acetylCoA arises from b-oxidation

Occurs when not enough oxaloacetate is available to react
with the large amounts of acetyl-CoA that could enter the
citric acid cycle

A situation like this can come about when an organism has
a high intake of lipids and a low intake of carbohydrates, but
there are also other possible causes, such as starvation and
diabetes

The reactions that result in ketone bodies start with the
condensation of two molecules of acetyl-CoA to produce
acetoacetyl-CoA

Acetoacetate is produced from acetoacetyl-CoA through
condensation with another acetyl-CoA to form -hydroxy-bmethylglutaryl-CoA (HMG-CoA), a compound we will see
again when we look at cholesterol synthesis
o A reduction reaction can produce hydroxybutyrate from acetoacetate.
o The other possible reaction is the spontaneous
decarboxylation of acetoacetate to give acetone

Ketosis – a pathological condition from excess of
acetoacetate, and consequently of acetone

18
First Steps in Fatty Acid Biosynthesis
In the cytosol, acetyl-CoA is carboxylated, producing
malonyl-CoA, a key intermediate in fatty acid biosynthesis


This reaction is catalyzed by the acetyl-CoA carboxylase
complex, which consists of three enzymes and requires
Mn21, biotin, and ATP for activity
Acetyl-CoA carboxylase consists of the three proteins biotin
carboxylase, the biotin carrier protein, and carboxyl
transferase
Two-Carbon Addition by Fatty Acid Synthase

The biosynthesis of fatty acids involves the successive
addition of two-carbon units to the growing chain

Two of the three carbon atoms of the malonyl group of
malonyl-CoA are added to the growing fatty acid chain with
each cycle of the biosynthetic reaction

Fatty acid synthase – a multienzyme complex located in the
cytosol, required in this biosynthetic reaction

The usual product of fatty acid anabolism is palmitate, the
16-carbon saturated fatty acid

There is a priming step in which one molecule of acetyl-CoA
is required for each molecule of palmitate produced

In this priming step, the acetyl group from acetyl-CoA is
transferred to an acyl carrier protein (ACP), which is
considered a part of the fatty acid synthase complex
The pathway of palmitate synthesis
from acetyl-CoA and malonyl-CoA
Fatty Acid Biosynthesis

The anabolism of fatty acids is not simply a reversal of the
reactions of b-oxidation

A first example of the differences between the degradation
and the biosynthesis of fatty acids is that the anabolic
reactions take place in the cytosol

The first step in fatty acid biosynthesis is transport of acetylCoA to the cytosol
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science

Comparison of Fatty Acid Degradation and Biosynthesis
Degradation
Product is acetyl-CoA
Biosynthesis
Precursor is acetyl-CoA
Malonyl-CoA is not involved; no
requirement for biotin
Malonyl-CoA is source of twocarbon units; biotin required
Oxidative process; requires NAD1
and FAD and produces ATP
Reductive process; requires
NADPH and ATP
Fatty acids form thioesters with
CoA-SH
Fatty acids form thioesters with
acyl carrier proteins (ACP-SH)
2

The conversion of phosphatidates to other phospholipids
frequently requires the presence of nucleoside
triphosphates, particularly cytidine triphosphate (CTP)
The role of CTP depends on the type of organism, because
the details of the biosynthetic pathway are not the same in
mammals and bacteria
o
2
Starts at carboxyl end (CH3CO2 )
Starts at methyl end (CH3CH2 )
Occurs in the mitochondrial
matrix, with no ordered
aggregate of enzymes
Occurs in the cytosol, catalyzed
by an ordered multienzyme
complex
b-Hydroxyacyl intermediates
have the l configuration
b-Hydroxyacyl intermediates
have the d configuration
Synthesis of Acylglycerols and Compound Lipids

Free fatty acids do not occur in the cell to any great extent;
they are normally found incorporated in triacylglycerols and
phosphoacylglycerols

The biosynthesis of these two types of compounds takes
place principally on the ER of liver cells or fat cells
(adipocytes)
Triacylglycerols

The glycerol portion of lipids is derived from glycerol-3phosphate, a compound available from glycolysis

In liver and kidney, another source is glycerol released by
degradation of acylglycerols

An acyl group of a fatty acid is transferred from an acyl-CoA
19
o
In bacteria, CTP reacts with phosphatidate to produce
cytidine diphospho-diacylglycerol (a CDP diglyceride). The
CDP
diglyceride reacts
with
serine to
form
phosphatidylserine.
Phosphatidylserine
is
then
decarboxylated to give phosphatidylethanolamine.
In eukaryotes, the synthesis of phosphatidylethanol-amine
requires two preceding steps in which the component parts
are processed. The first of these two steps is the removal by
hydrolysis of the phosphate group of the phosphatidate,
producing a diacylglycerol; the second step is the reaction of
ethanolamine phosphate with CTP to produce pyrophosphate
(PPi) and cytidine diphosphate ethanolamine (CDPethanolamine. The CDP-ethanolamine and diacylglycerol
react to form phosphatidylethanolamine
Sphingolipids

The structural basis of sphingolipids is not glycerol but
sphingosine, a long-chain amine

The precursors of sphingosine are palmitoyl-CoA and the
amino acid serine, which react to pro- duce
dihydrosphingosine

Reaction of the amino group of sphingosine with another
acyl-CoA to form an amide bond results in an Nacylsphingosine, also called a ceramide.

Ceramides in turn are the parent compounds of
sphingomyelins, cerebrosides, and gangliosides
Cholesterol Biosynthesis
From Acetyl-CoA to Cholesterol

The ultimate precursor of all the carbon atoms in
cholesterol and in the other steroids that are derived from
cholesterol is the acetyl group of acetyl-CoA

The involvement of isoprene units is a key point in the
biosynthesis of steroids

Six isoprene units condense to form squalene, which
contains 30 carbon atoms

Finally, squalene is converted to cholesterol, which
contains 27 carbon atoms; squalene can also be converted
to other sterols
Pathways for the biosynthesis of triacylglycerols
Phosphoacylglycerols

Phosphoacylglycerols (phosphoglycerides) are based on
phosphatidates, with the phosphate group esterified to
another alcohol, frequently a nitrogen-containing alcohol
such as ethanolamine
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
HMG-CoA in Cholesterol Biosynthesis

This reaction is catalyzed by the enzyme
hydroxymethylglutaryl-CoA synthase; one molecule of
CoA-SH is released in the process

In the next reaction, the production of mevalonate from
hydroxymethylglutaryl-CoA is catalyzed by the enzyme
hydroxymethylglutaryl-CoA reductase (HMG-CoA
reductase)

A carboxyl group, the one esterified to CoA-SH, is reduced
to a hydroxyl group, and the CoA-SH is released

This step is inhibited by high levels of cholesterol and is the
major control point of cholesterol synthesis

It is also a target for drugs to lower cholesterol levels in
the body

Mevalonate is then converted to an isoprenoid unit by a
combination of phosphorylation, decarboxylation, and
dephosphorylation reactions

Condensation of isoprenoid units then leads to the
production of squalene and, ultimately, cholesterol
20
The synthesis of bile acids
from cholesterol


Cholesterol is the precursor of important steroid hormones
– serve as signals from outside a cell that regulate metabolic
processes within a cell
Steroids are best known as sex hormones
The synthesis of steroid
hormones from cholesterol




Cholesterol is synthesized from squalene via lanosterol

Cholesterol as Precursor to Other Steroids

After cholesterol is formed, it can be converted to other
steroids of widely varying physiological function

The smooth ER is an important site for both the synthesis of
cholesterol and its conversion to other steroids

Most of the cholesterol formed in the liver, which is the
principal site of cholesterol synthesis in mammals, is
converted to bile acids – aid in the digestion of lipid droplets
by emulsifying them and rendering them more accessible to
enzymatic attack
Pregnenolone is formed from cholesterol, and
progesterone is formed from pregnenolone
Progesterone is a sex hormone and is a precursor for other
sex hormones, such as testosterone and estradiol (an
estrogen)
Cortisone is an example of glucocorticoids, a group of
hormones that play a role in carbohydrate metabolism, as
well as in the metabolism of proteins and fatty acids.
Mineralocorticoids constitute another class of hormones
that are involved in the metabolism of electrolytes,
including metal ions (“minerals”) and water
Aldosterone is an example of a mineralocorticoid
The Role of Cholesterol in Heart Disease: LDL and HDL

Atherosclerosis is a condition in which arteries are blocked
to a greater or lesser extent by the deposition of cholesterol
plaques, which can lead to heart attacks

Both diet and genetics are instrumental in the development
of atherosclerosis
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science




21
higher body fat; also stimulates production of leptin in
adipocytes
o Leptin – another protein hormone, consisting of 146
amino acid residues; stimulates breakdown of lipids
and inhibits fatty acid production; its production
increases when fat deposits in adipocytes become
larger; when the signal of higher leptin levels reaches
the central nervous system, the result is decrease in
appetite; high levels of leptin are interpreted as
overfeeding, while low levels of leptin in the
bloodstream are interpreted as starvation
Cholesterol must be packaged for transport in the
bloodstream; several classes of lipoproteins are involved in
the transport of lipids in blood
Low-density lipoproteins (LDLs) and high-density
lipoproteins (HDLs) will play the major role in our discussion
of heart disease
LDL is the major player in the development of
atherosclerosis
Unlike LDL, which transports cholesterol from the liver to
the rest of the body, HDL transports it back to the liver for
degradation to bile acids
Carbohydrates
Carbohydrates referred to compounds of the general formula
Cn(H2O)n (only the simple sugars or monosaccharides fit this
formula)
Oligosaccharides (Greek oligos) are formed when a few
monosaccharides are linked; under this is

Disaccharides— formed by linking two monosaccharide
units by glycosidic bonds
Polysaccharides (Greek polys) are formed when many
monosaccharides are bonded together
Loss of one H2O for each new linked form – adds monosaccharide
units to a growing carbohydrate
Carbohydrate Function


The formation of atherosclerosis, depicting the growth of an
atherosclerotic plaque in a coronary artery
Hormonal Control of Appetite




Hormones from the brain, stomach, intestines, pancreas,
and adipose tissue all play a role in stimulating and in
repressing appetite
In the part of the brain called the arcuate nucleus, two sets
of neurons play a role:
1. The neurons that stimulate eating are called the
NPY/AgRP-producing neurons because they produce
neuropeptide Y (NPY), which, in turn, stimulates other
neurons that eventually lead to increased appetite
2. The neurons that tend to inhibit eating produce
melanocortins, another class of peptide hormones,
which also stimulate other neurons
The peptide hormones ghrelin and cholecystokinin are the
main regulators of short-term effects
o Ghrelin – produced in the stomach, primarily when the
stomach is empty; its production is a hunger signal and
falls off as food is eaten
o Cholecystokinin – the signal for satiety, and so its
effect is the opposite of that produced by ghrelin
Insulin and leptin are the hormones most deeply involved in
long-term control of eating behavior
o Insulin – small protein consisting of 51 amino acid
residues, is produced in the b-cells of the pancreas;
stimulates glucose uptake into many tissues, including
adipose tissue; higher insulin levels correlate with


Major energy sources
Many commonly encountered carbohydrates are
polysaccharides
Oligosaccharides play a key role in processes that take
place on the surfaces of cells, particularly in cell-cell
interaction and immune recognition
Polysaccharides are essential structural components of
several classes of organisms;
Carbohydrates Structure
Monosaccharides (simple sugars) can be a:

Polyhydroxy aldehyde (aldose) – most common sugarsd
aldoses are classified according to the number of carbon atoms:
1. aldotriose (simplest, three carbons ex. Glyceraldehyde)
2. aldotetrose (photosynthesis & other metabolic pathways,
four carbons)
3. aldopentose (one occurs in the structure of RNA five
carbons)
4. aldohexose (most abundant in nature, six carbons)

Polyhydroxy ketone (ketose)
ketoses are also classified according to the number of carbon
atoms:
1. ketotriose (simplest, three carbons ex. dihydroxyacetone)
2. ketotetrose (photosynthesis & other metabolic pathways,
four carbons)
3. ketopentose (one occurs in the structure of DNA, five
carbons)
4. ketohexose (most abundant in nature, six carbons)
Stereoisomers (optical isomers) – molecules that differ from each
other only in their configuration (three-dimensional shape)
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
Chiral (asymmetric) carbon atom – is the usual source of optical
isomerism
Enantiomers – mirror-image, non-superimposable stereoisomers
Note: Dihydroxyacetone does not contain a chiral carbon atom and
does not exist in nonsuperimposable mirror-image forms
Configuration - three-dimensional arrangement of groups around a
chiral carbon atom; arrangement of the OH group

The D, L system is used to denote stereochemistry
Dashed wedges – represents bonds directed away from the viewer
Solid wedges – represents bonds directed oppositely, toward the
viewer and out of the plain of the paper
22
hydroxyl group is on the LEFT of the highest-numbered chiral carbon
in the L configuration (use the picture above as basis)
For glucose and fructose, since both of them are hexoses, the chiral
carbon with the highest number is C-5. It will be the basis if the
configuration is L or D. Aldose is always on the right side, while
ketose naman is always on the left side
Diastereomers – nonsuperimposable, non-mirror image
stereoisomers
Applicable only to aldotetroses, aldopentoses, aldohexoses since
they have a lot of stereoisomers. Like the aldotetroses for example.
If you’ll look at the chart [Figure 1] there’s two (2) aldotetrose which is
Erythrose and Threose so meaning it has four (4) stereoisomers (D,L
Erythrose and D,L Threose). The D Erythrose and D Threose they
have the same configuration (or the arrangement of the –OH group)
in C-3 but when it comes to the other chiral carbon it’s different.
Also, these two D are not enantiomers of each other rather they are
diastereomers.
with that being said..



The possibilities for stereoisomerism increase as the
number of carbon atoms increases.
The designation of the configuration as L or D depends on
the arrangement at the chiral carbon with the highest
number
D sugars predominate in nature than L sugars.
Fischer Projection Method
L Erythrose is the enantiomer of D Erythrose
L Threose is the enantiomer of D Threose
L Erythrose is the diastereomers of both D, L Threose
L Threose is the diastereomers of both D,L Erythrose
Epimers – stereoisomers that differ only in configuration around one
chiral carbon atoms
example: D Erythrose and D Threose are epimers
D sugars – predominate in nature
Glucose – an ubiquitous (found everywhere) energy source
Ribose – plays an important role in the structure of nucleic acids
Cyclic Forms
Cyclization takes place as a result of interaction between
the functional groups on distant carbons; Sugars with five or six
carbon atoms normally exist as cyclic molecules
1.
2.






two-dimensional perspective of the molecular structure;
named after the German chemist Emil Fischer
Carbon atoms are numbered in sequence from the “top”
carbon.
Bonds written vertically on the 2D paper represent bonds
directed behind the paper in 3D
Bonds written horizontally on the 2D paper represent
bonds directed in front of the paper in 3D
In Aldoses, the most highly oxidized carbon is written at
the “top” and is C-1
In Ketoses, the most highly oxidized carbon is written next
to the top and is C-2
Hemiacital (aldohexoses) – interaction between C-1 and C-5
Hemiketal (aldohexoses) -- interaction between C-2 and C-5
Anomeric Carbon – the carbonyl carbon that becomes a new chiral
center
Two forms (anomers of each other):

Designated α

Designated β
Free carbonyl species can readily form either the α- or β-anomer,
and the anomers can be converted from one form to another
through the free carbonyl species

In some biochemical molecules, any anomer of a given
sugar can be used, but, in other cases, only one anomer
occurs
Fischer projection of the D configuration, the hydroxyl group is on
the RIGHT of the highest-numbered chiral carbon, whereas the
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
Fischer Projection – useful for describing the stereochemistry of
sugars
23
Chair conformation - Widely shown in organic chemistry text books

Useful in discussion of molecular recognition

Used by Organic chemists
Note: Chair conformation and Haworth Projection are alternative
ways of expressing the same information
Haworth Projection – gives a realistic picture of the bonding
situation in cyclic forms; accurately represents the overall shape of
the molecules

Approximations

Useful shorthand for the structures of reactants and
products

Used by Biochemist

Cyclic structures of sugars are shown in perspective
drawing as planar five- (Furanose) or six- (Pyranose)
membered rings viewed nearly edge on
Note: Any group that is written to the right of the carbon in a Fischer
projection has a downward direction in a Haworth projection; any
group that is written to the left in a Fischer projection has an
upward direction in a Haworth Projection
Reactions of Monosaccharides
Redox reactions of Simple Sugars
Oxidation (Lose) – loses hydrogen electron, gains O2; occurs when
an atom molecule or ion loses one or more electrons in a chemical
reactions

Oxidation of sugars provides energy for organisms to carry
out their life processes

The highest yield of energy from carbohydrates occurs
when sugars are completely oxidized to CO2 and H2O in
aerobic processes

Oxidation processes used to identify sugars

Basis of a test for the presence of aldoses – Aldehyde
groups can be oxidized to give the carboxyl group
(characteristic of acids). When aldehyde is oxidized, some
oxidizing agent must be reduced
Reduction (Gain) – gains hydrogen electron, loses O2; involves the
gaining of electrons by one of the atoms involved in the reaction
between two chemicals



Reverse of complete oxidation of sugars is the reduction
CO2 and H2O of sugars (photosynthesis)
Aldoses reducing sugars (Ketoses can be reducing sugars
because they isomerize to aldoses
Reducing sugars are sugars that have a free carbonyl
group, one that can react with an oxidizing agent
Some important reduced sugars:

Doexy sugars – a hydrogen atom is substituted for one of
the hydroxyl groups of the sugar
o L-fucose (L-6-deoxygalactose) – found in the
carbohydrate portion of some glycoprotein
o D- deoxyribose
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
Glycoprotein (glykos is Greek for “sweet”) – indicated that these
substances are conjugated proteins that contain some carbohydrate
group in addition to the polypeptide chain
24
Glycosidic linkage (R’—O—R) – is formed when a sugar hydroxyl
group (ROH) bonded to an anomeric carbon to react with another
hydroxyl (R’—OH)

Not an ether (glycosides can be hydrolyzed to the original
alcohols

Involves the anomeric carbon of the sugars in its cyclic
form

Hemiacetal carbon can react with an alcohol such as
methyl alcohol to give a full acetal or Glycoside. The newly
formed bond is a glycosidic bond

Can take various forms:
o The anomeric carbon of one sugar can be bonded to
any one of the –OH groups on a second sugar to form
an α- or β-glycosidic linkage
o –OH groups are numbered so that they can be
distinguished (follows the carbon atoms)
Two types of reagent used to detect the presence of reducing sugars:
1. Tollens reagent – uses silver ammonia complex ion
Ag(NH3)2+ as oxidizing agent; A silver mirror is deposited
on the wall nof the test tube if a reducing sugar is present
2. Gluxose oxidase- an enzyme used in the modern method
to detect the presence of glucose but not other reducing
sugars
Alditols – one of the polyhydroxy alcohols which is a resulting
compound when the carbonyl group of a sugar is reduced to
hydroxyl group
Example: xylitol and sorbitol (derivatives of sugar xylulose and
sorbose–sweeteners in sugarless chewing gums and candy)
Glycosides – a compound in which one or more sugars is bonded to
another molecule
o Furanosides glycoside involving furanose
o Pyranosides glycosides involving pyranose
o Internal anomeric carbons in oligosaccharides are not
free to give the test for reducing sugars. Only if the
end residue is a free hemiacetal rather than a
glycoside will there be a positive test for reducing
sugars
Sugar Esters and Ethers
Hydroxyl groups of sugars reacts with acids and derivatives of acids
to form esters
Phosphate Esters – particularly important; the suual intermediates
in the breakdown of carbohydrates to provide energy

Formed by transfer of a phosphate group from ATP to ADP
(important in the metabolism of sugars)

Glycosidic bonds – between monosaccharide units, it is the basis for
the formation of oligosaccharides and polysaccharide

When oligosaccharides and polysaccharides form as a
result of glycosidic bonding, their chemical natures depend
on which monosaccharides are linked together and also on
the particular glycosidic bond formed
Two ways in which α-D-glucose molecules can be linked together:

α (1 → 4)
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science

25
α (1 → 6)
β-D-glucose molecules: β, β (1 → 1)
Some Important Oligosaccharides
Three of the most important oligosaccharides (that are
disaccharides):

Sucrose

Lactose

Maltose
Variation in glycosidic linkages forms both linear and branched-chain
polymers:
1. Linear polymers – If the internal monosaccharide residue
form only two glycosidic bonds
2.
Branched-chain polymers – if some internal residues can
form three glycosidic bonds
Amino Sugars
Interesting class of compounds related to the
monosaccharides. Sugars with substituted amino group as part of it
structure
An amino group (—NH2) or one of its derivatives is

subtituted for the hydroxyl group of the parent sugar
o N-acetyl amino sugars, the amino group itself carries
an acetyl group (CH3—CO—) as a substituent
o N-acetyl-β-D-Glucosamine and N-acetyl-β-muramic
acid (has an added carboxylic acid) – components of
bacterial cell walls
Note: the D configuration and the β-anomeric form are so common
that we need not to specify them (if L or D series of configuration or
α-anomer or β-anomer). This type of shorthand is the usual practice
with β-D-glucose
Sucrose formed when glucose and fructose are bonded together

Common table sugar extracted from sugarcane and sugar
beets

Not a reducing sugar because both anomeric groups are
involved in the glycosidic linkage

When animals consume sucrose, it is hydrolyzed to glucose
and fructose which are then degraded by metabolic
processed to provide energy

Excess consumption of sucrose can contribute to health
problems - led to a search for other sweetening agents:
o Fructose – sweeter than fructose; smaller amount (by
weight) of fructose than sucrose can produce the
same sweetening effect with fewer calories

Artificial sweeteners suspected of having harmful side
effects:
o Saccharin – found to cause cancer in laboratory
animals. Applicability of these results to human
carcinogenesis has been questioned by some
o Aspartame (NutraSweet) – suspected of causing
neurological problems (especially in individuals whose
metabolism cannot tolerate phenylalanine)
o Sucralose (derivative of sucrose) – not metabolized
by the body; does not provide calories. Anecdotal
evidence indicate that it is a safe sugar substitute
Differs from sucrose in two ways:
1. Three if the hydroxyl groups have been replaced
with three chlorine atoms. Bonded to carbon
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
atoms 1 and 6 of the fructose moiety and to
carbon atom 4 of the galactose moiety;
2. The configuration at carbon atom 4 of the sixmembered pyranose ring of glucose has been
inverted, producing galactose derivative
Lactose made up of β-D-galactose and D-glucose

A reducing sugar because the group at the anomeric
carbon of glucose portion is not involved in a glycosidic
linkage. It is free to react with oxidizing agents

The manner in which lactose is metabolized by the body
can have important health implications (Lactose
Intolerance)
Maltose obtained from the hydrolysis of starch

Differs from cellobiose only in the glycosidic linkage

Mammals can digest maltose

Used in other beverages (malted milk)
Cellobiose obtained from the hydrolysis of cellulose

Mammals cannot digest cellobiose



26
Individual polysaccharide chains are hydrogen-bonded
together, giving plant fibers their mechanical strength
Animals lack the enzyme cellulases (hydrolyzes cellulose to
glucose)
o Attacks the β-linkages between glucoses which is
common to structureal polymers
o Found in certain bacteria, including bacterias that
inhabit the digestive tracts of insects (termites, cattle,
and horses)
Plays an important role as a component of dietary fiber
Branched Homopolysaccharides of Glucose
Polysaccharides, such as starches, serve as vehicles for
storage of glucose
Starches (plants) are polymers of α-D-glucose that occur in plant
cells (usually as starch granules in the cytosol)

There’s an α-linkage in contrast with the β-linkage of the
cellulose

The types of starches can be distinguished from one
another by their degrees of chain branching
o Amylose – a linear polymer of glucose; with all the
residues linked together by α (1 → 4) bonds
Structures and Functions of Polysaccharides
Polysaccharides many monosaccharides linked together

Polysaccharides that occur in organisms usually composed
of a very few types of monosaccharide components

Complete characterization of polysaccharides includes
specification of which monomers are present and, if
necessary, the sequence of monomers

Requires the type of glycosidic linkage be specified
(nature of the linkage determines functions)
o Polysaccharide with β-glycosidic linkages – Cellulose
and Chitin
o Polysaccharide with α-glycosidic linkages –
Starch and Glycogen
Homopolysaccharide – a polymer that consists of only one type of
monosaccharide
Heteropolysaccharide – a polymer consists of more than one type of
monosaccharide (frequently, only two types of molecules occur in a
repeating sequence
o


Linear Homopolysaccharides of Glucose
Cellulose is the major structural components of plants (especially of
wood and plant fibers)

Linear homopolysaccharide of β-D-Glucose and allresidues
are linked in β (1 → 4) glycosidic bonds
Amylopectin – branched chain polymer; with the
branches starting at α (1 → 6) linkages along the
chain of α (1 → 4) linkages
o Branch points occur about every 25 residues
Because starches are storage molecules, there must be a
mechanism for releasing glucose from starch when in the
organism needs energy
Both plants and animals contain enzymes that hydrolyze
starches
o α-amylase – an endoglysidase which can hydrolyze a
glycosidic linkage anywhere along the chain to
produce glucose and maltose.
o β-amylase – an exoglysidase that cleaves from the
non-reducing and of the polymer; Maltose (dimer of
glucose) is theproduct of reaction
Amylose can be completely degraded to glucose and maltose by the
two amylases; However, Amylopectin is not completely degraded
because the branch linkages are not attacked

Debranching enzymes occur in both plants and animals;
they degrade the α (1 → 6) linkages
o Combined with the amylases, they can contribute to
the complete degradation of both forms of starch
o Plays a role in the complete breakdown of glycogen
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
Glycogen (animals) is a branched-chain polymer of α-D-glucose;
similar to the amylopectin fraction of starch

Consist of a chain of α (1 → 4) linkages with a α (1 → 6)
linkages at the branch points (similar to Amylopectin)

More highly branched than Amylopectin

Branch points occur about every 10 residues in glycogen

Average chain length is 13 glucose residues, and 12 layers
of branching





Glycogenin - at the heart of every glycogen molecule
Glycogen found in animal cells in granules similar to the
starch granules in plant cells
Glycogen granules are observed in well-fed liver and
muscle cells, but they are not seen in other cell types
(brain and heart cells under normal condition)
When the organism needs energy, various degradative
enzymes remove glucose units:
o Glycogen Phosphorylase – it cleaves one glucose at a
time from the non-reducing end of a branch to
produce glucose-1-phosphate, which then enter the
metabolic pathways of carbohydrate breakdown
Number of branch points is significant for two reasons (for
mammals):
1. More branched polysaccharide is more water
soluble
o Glycogen-storage diseases caused by lower-thannormal levels of branching enzymes. Glycogen
products resemble starch and can fall out of
solution, forming glycogen crystals in the
muscles and liver
2. When an organism needs energy quickly, the
glycogen phosphorylase has more potential targets if
there are more branches, allowing a quicker
mobilization of glucose
Linear Homopolysaccharides of Glucose Derivatives
27
(insects and crustaceans), and it also occurs in cell
walls of algae, fungi, and yeasts
Heteropolysaccharides
Heteroploysaccharides are major components of bacterial cell walls

Prokaryotic cells walls – polysaccharides are cross-linked
by peptides

Repeating unit of the polysaccharide consists of two
residues held together by β (1 → 4) glycosidic links

Two monomers:
1. N-acteyl-D-glucosamine
2. N-acetylmuramic acid – found only in prokaryotic cell
walls; does not occur in eukaryotic cell walls
Differs by the substitution of a lactic acid side chain [—O—
CH(CH3)—COOH] for the hydroxyl group (—OH) on carbon 3




Cross-links in bacterial cell walls consist of small peptides
Extensive cross-linking produces a three-dimensional
network of considerable mechanical strength (which is
why bacterial cell walls are extremely difficult to disrupt)
Peptidoglycan – material that results from the crosslinking of polysaccharides by peptides
o So named because it has both peptide and
carbohydrate components
Plant cell walls consists of
o Cellulose – largely; important structural material in
plants
o Pectin a polymer made up mostly of D-galacturonic
acid, a derivative of galactose in which the hydroxyl
group on carbon C-6 has been oxidized to a carboxyl
group
o Extracted from plants; has commercial
importance in the food processing industry as a
gelling agent (yogurt, fruit preserves, jams, and
jellies)
Chitin similar to cellulose in both structure and function


A linear homopolysaccharide with all the residues linked in
β (1 → 4) glycosidic bonds
Differs from cellulose in the nature of monosaccharide unit
o The monomer in cellulose is β-D-glucose
o The monomer in Chitin is N-acetyl- β-D-glucosamine
(differs from glucose only in the substitution of the Nacetylamino group (—NH—CO—CH3) for the hydroxyl
group (—OH) on carbon
o Plays a structural role and has a fair amount of
mechanical strength because the structural
component of the exoskeletons of invertebrates
o
Lignin – major nonpolysaccharide component
especially in woody plants (Latin lignum, “wood”)
o Polymer of coniferyl alcohol, and it is a very
tough and durable material
Contain comparatively little peptide or protein compared to bacterial
cell walls
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
28
Specific Roles in Connective Tissues
Glycoprotein
Glycosaminoglycans are a type of polysaccharide based on a
repeating disaccharide in which one of the sugars is an amino sugar
and at least one of them has a negative charge owing to the
presence of sulfate group or carboxyl group

Involved in a wide variety of cellular functions and tissues
Glycoprotein contains carbohydrate residues in addition to the
polypeptide chain

Important glycoproteins involved in the immune response:
Antibodies – binds to and immobilize antigens (substance
attacking the organisms)

Plays a role as Antigenic determinants – the portions of an
antigenic molecule that antibodies recognize and to which
they bind
o Found in human blood groups:

A – contains L-fucose; N-Acetylgalactosamine is
found at the non-reducing end of the
oligosaccharide in the type-A blood group
antigen

B – contains L-fucose; α-D-galactose found at the
non-reducing end of the oligosaccharide in the
type-B blood group antigen

AB – contains L-fucose; neither

O – contains L-fucose N-Acetylgalactosamine and
α-D-galactose are present
Most common Glycosaminoglycans:
1. Heparin is a natural anticoagulant that helps prevent
blood clots
2.
Hyaluronic acid is a component of the vitreous humor of
the eye and of the lubricated fluid of joints
The distinctions between the groups depend in the oligosaccharide
portions of the glycoprotein on the surfaces of the blood cells called
erythrocytes
3.
Chondroitin sulfates and Keratan sulfate are components
of connective tissues



4.
5.
Glucosamine sulfate and chondroitin sulfate used to help
repair frayed or otherwise damaged cartilage, especially in
knees
Dermatan sulfate is a glycosaminoglycan found mostly in
skin, but also in blood vessels, heart valves, tendons, and
lung
Plays an important role in eukaryotic cells membranes
The sugar portions are added to the protein as it passes
through the Golgi on its way to the cell surface
Proteoglycans glycoproteins with an extremely high
carbohydrate content (85%-95% by weight) are classified
as proteins
o Constantly being synthesized and broken down
o If there is lack of the lysosomal enzymes that
degrade them, proteoglycans accumulate, with
tragic consequences

Hurler’s Syndrome – material that
accumulates includes large amounts of
amino sugars (leads to skeletal deformities,
severe mental retardation, and death in
early childhood)
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
Stereochemical relationships among monosaccharides.
The linear form of D-glucose undergoes an intramolecular reaction to form a cyclic hemiacetal
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
29
Biochemistry for Medical Laboratory Science
30
Glycolysis
THE OVERALL PATHWAY OF GLYCOLYSIS
Step 3: Phosphorylation of fructose-6-phosphate to give fructose1,6-bisphosphate (ATP is the source of the phosphate group)
Glycolysis (Glycolytic Pathway)

Also known as Embden-Meyerhoff pathway

First stage pf glucose metabolism in organisms from bacteria
to human

Plays a key role in the way organisms extract energy from
nutrients
Step 4: Cleavage of fructose-1,6-bisphosphate to give two 3-carbon
fragments, glyceraldehyde-3-phosphate and dihydroxyacetone
phosphate
Pyruvate: A Key Intermediate in Glycolysis
Step 5: Isomerization of dihydroxyacetone phosphate to give
glyceraldehyde-3-phosphate
Several Fates of Pyruvate:
Step 6: Oxidation (and phosphorylation) of glyceraldehyde-3phosphate o give 1,3-bisphosphoglycerate
Step 7: Transfer of phosphate group from 1,3-bisphosphoglycerate
to ADP (Phosphorylation of ADP to ATP) to give 3-phosphoglycerate
Step 8: Isomerization of 3-phosphoglycerateto give 2phosphoglycerate
Step 9: Dehydration of 2-phosphoglycerate to give
phosphoenolpyruvate
Step 10: Transfer of a phosphate group from phosphoenolpyruvate
to ADP (phosphorylation of ADP to ATP) to give pyruvate
1.
Aerobic Metabolism – Pyruvate loses CO2, the remaining
two carbon atoms become linked to coenzyme A as an acetyl
group to form acetyl CoA → TCA cycle
2. Anaerobic Metabolism

Organism capable of alcoholic fermentation – Pyruvate
loses CO2 produces acetaldehyde → reduced to produce
ethanol

Anaerobic Glycolysis– common fate of pyruvate;
reduction to lactate
Glucose to product is (oxidation reaction)

Requires an accompanying reduction reaction: NAD+ is
converted to NADH
Summary of breakdown of glucose to pyruvate:
Note: only one of the 10 steps in this pathway involves an electrontransfer reaction.
The Ten Reactions of Glycolysis
CONVERSION OF SIX-CARBON GLUCOSE TO THREE-CARBON
GLYCERALDEHYDE-3-PHOSPHATE
First steps of the glycolytic pathway that prepares for the
electron transfer and phosphorylation of ADP

Makes use of the free energy of hydrolysis of ATP

Preparation phase of Glycolysis
Step 1: Glucose is phosphorylated to give glucose-6-phosphate
(Endergonic)
Hydrolysis of ATP (Exergonic)
Check figure 17.3 for the reaction sequence of the Glycolytic
pathway
Sum of the two reactions = Exergonic
The Ten Reactions of Glycolysis
Step 1: Phosphorylation of glucose to give glucose-6-phosphate (ATP
is the source of the phosphate group)
Step 2: Isomerization of glucose-6-phosphate to give fructose-6phosphate
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
31
∆G° = calculated under standards states with the concentration of all
reactants and products at 1M except hydrogen ion
In the free-energy change, the number varies depending
on cell type and metabolic state

Typical value for this reaction: -33.9 kJ mol-1 or -8.12 kcal
mol-1
Check table 17.1 for the ∆G°' and ∆G values for all the reactions of
anaerobic glycolysis and erythrocytes



Phosphorylation of glucose illustrate the use of chemical
energy originally produced by the oxidation of nutrients
and ultimately trapped by phosphorylation of ADP to ATP
Chemical energy of nutrients is released by oxidation and
is made available for immediate use on demand by being
trapped as ATP
Enzyme: Hexokinase
o Kinase – applied to the class of ATP-dependent
enzymes that transfer a phosphate group from ATP to
a substrate
o Substrate can by any one of a number of hexoses (i.g.
glucose, fructose, and mannose)
o
Glucose-6-phosphate inhibits the activity of hexokinase
(control
o Some organisms or tissues contain multiple isozymes
of hexokinase
o Glucokinase – one isoform of hexokinase found in the
human liver; lowers blood glucose levels after one has
eaten a meal

Requires much high substrate level to achieve
saturation

↑ glucose levels – liver can metabolize glucose via
glycolysis preferentially over other tissues

↓ glucose levels – hexokinase is still active in all
tissues
o Large conformation change takes place in hexokinase
when substrate is bound

X-ray crystallography: in the absence of substrate,
two lobes of the enzyme that surround the binding
site are quite far apart
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science

32
When glucose is bound, the two lobes move closer
together, and the glucose becomes almost
completely surrounded by protein (consistent with
the induced-fit theory of enzyme)
Note: in all kinases, a cleft closes when substrate is bound

Enzyme: phosphofructokinase – key allosteric control
enzyme in glycolysis
o A tetramer that is subject to allosteric feedback
regulation
o There are two types of subunits: M and L

Can combine into tetramers to give different
permutations (M4, M3L, M2L2, ML3, and L4)

Isozymes—combination of subunits; multiple
forms of an enzyme that catalyze the same overall
reaction but have subtle physical and kinetic
differences

Differs slightly in amino acid composition; two
enzymes can be separated from each other by
electrophoresis
Tetramic forms in:
a. Muscle - M4
b. Liver - L4
c. RBC – several combinations can be found
Note: individuals who lack the gene that directs the synthesis of the
M form of the enzyme can carry on glycolysis in their livers but
experience muscle weakness due to the lack of enzyme in the
muscle
Step 2: Glucose-6-phosphate isomerizes to give fructose-6phosphate






Enzyme: Glucose phosphate isomerase
C-1 aldehyde group of glucose-6-phosphate is reduced to a
hydroxyl group
C-2 hydroxyl group is oxidized to give the ketone group of
fructose-6-phosphate (no net oxidation or reduction)
Phosphorylated forms: glucose-6-phosphate (aldose;
fructose-5-phosphate (ketose)
Step 3: Fructose-6-phosphate is further phosphorylated, producing
fructose-1,6-bisphosphate




Highly exergonic and irreversible
Endergonic reaction of phosphorylation of Fructose-6phosphate is coupled to the exergonic reaction of
hydrolysis of ATP; Overall reaction = Exergonic
This reaction is the one in which the sugar is committed to
glycolysis
Glucsoe-6-phosphate and fructose-6-phosphate can play
roles in other pathways
fructose-1,6-bisphosphate does not play any roles in other
pathways; no other pathway is available and the molecule
must undergo the rest of the reactions of glycolysis
When rate of the phosphofructokinase reaction is
observed at varying concentrations of substrate, sigmoidal
curve typical of allosteric enzymes is obtained
o ATP is an allosteric effector in the reaction
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science

↑ ATP depress the rate of the reaction – a good deal
of chemical energy is immediately available from
hydrolysis of ATP
 ↓ ATP stimulate the reaction
 The presence of ATP inhibits the glycolytic pathway
o Fructose-1,6-bisphosphate more potent allosteric
effector of phosphofructokinase
Step 4: Fructose-1,6-bisphosphate is split into two three-carbon
fragments




Enzyme: aldolase – catalyzes reverse aldol condensation of
Fructose-1,6-bisphosphate
Cleavage reaction is the reverse of an aldol condensation
In the enzyme isolated from most animal sources, the
basic side chain of an essential lysine residue plays the key
role in catalyzing this reaction
Thiol group of a cysteine also acts as base
Step 5: The dihydroxyacetone phosphate is converted to
glyceraldehyde-3-phosphate




Enzyme: triosephosphate isomerase
Second molecule of glyceraldehyde-3-phosphate (first
molecule has already been produced by the aldolase
reaction)
Glucose has now been converted to two molecules of
glyceraldehyde-3-phospahte (triose)
∆G value (under physiological conditions): +2.41 kJ mol -1
or +0.58 kcal mol-1; slightly positive
Note: we might think that the reaction would not occur and
glycolysis would be halted but glycolysis is composed of many
reactions that have very negative ∆G values that can drive the
reaction to completion
GLYCERALDEHYDE-3-PHOSPHATE IS CONVERTED TO PYRUVATE
Payoff phase of Glycolysis (ATP is produced instead of used)
In the rest of the pathway, two molecules of each of the
three-carbon compounds take part in evert reaction for each original
molecule
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
33
Biochemistry for Medical Laboratory Science

34
Phosphate ion attacks the thioester, forming a
mixed anhydride of the carboxylic and phosphoric
acids (high energy compound)
Step 6: Glyceraldehyde-3-phosphate is oxidized to 1,3bisphosphogylcerate


The characteristic reaction of glycolysis
Enzyme: glyceraldehyde-3-phosphate dehydrogenase –
important enzyme in both glycolysis and gluconeogenesis
o One of the NADH-Linked dehydrogenase class of
similar enzymes
o X-ray crystallography: overall structures are not
strikingly similar, but the structure of the binding site
for NADH is quite similar in all these enzymes


Involves the addition of a phosphate group to
glyceraldehyde-3-phosphate and an electron-transfer
reaction from glyceraldehyde-3-phosphate to NAD+
The half reaction of oxidation is that of an aldehyde to a
carboxylic acid group (H2O can be considered to take part
in the reaction)
The half reaction of reduction is that of NAD+ to NADH
The overall redox reaction



o
Molecule of this enzyme is a tetramer consisting four
identical subunits

Each subunit binds one molecule of NAD+

Each subunit contains an essential cysteine residue
o Key intermediate: Thioester involving the cysteine
residue is the

R – portions of the molecule other than the aldehyde and
carboxylic acid groups
Oxidation reaction is exergonic under standard conditions
(part of the overall reaction)
∆G°' = -43.1 kJ mol-1 = -10.3 kcal mol -1
Phosphate group linked to the carboxyl group does not
form an ester (ester linkages requires an alcohol and an
acid)
Carboxylic acid group and phosphoric acid form a mixed
anhydride of two acids by loss of water

Substances involved in the reaction are in the ionized form
appropriate at pH 7
Note: ATP and ADP do not appear in the equation. The source of the
phosphate group is phosphate ion itself rather than ATP
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science



Phosphorylation reaction is endergonic under standard
conditions
∆G°' = 49.3 kJ mol-1 = 11.8 kcal mol -1
Overall reaction, including electron transfer and
phosphorylation
Standard free-energy change for the overall reaction: sum
of the values for the oxidation and phosphorylation
Oxidation
35

a phosphate group is transferred from 1,3bisphosphogylcerate to a molecule of ADP, producing ATP
(first two reactions of the glycolytic pathway)
Note: 1,3-bisphosphogylcerate, a substrate transferred a phosphate
group to ADP typical in substrate-level phosphorylation

Substrate-level phosphorylation a reaction in which the
source of phosphorus is inorganic phosphate ion, not ATP
o It is to be distinguished from oxidative phosphorylation
in which transfer of phosphate groups is linked to
electron-transfer reactions in which oxygen is the
ultimate electron acceptor
o Requirement: standard free-energy of the hydrolysis
reaction is more negative than that for hydrolysis of
the new phosphate compound being formed

Standard free-energy of 1,3bisphosphogylcerate: -49.3 kJ mol-1

Standard free-energy of hydrolysis of ATP: -30.5
kJ mol-1
Note: change the sign of the free-energy change when the reverse
reaction occurs
Net reaction:
Products: two molecules of ATP for each molecule of glucose
that enters the glycolytic pathway
Phosphorylation
Step 8: The phosphate groups is transferred from carbon 3 to carbon
2 of the glyceric acid backbone

Overall reaction: slightly endergonic

Production of ATP requires a high-energy compound as
starting material

Step 7: One of the two reactions in which ATP is produced by
phosphorylation of ADP

Enzyme: phosphoglycerate kinase
Enzyme: phosphoglycermutase – catalyzes the
isomerization of 3-phosphoglycerate to 2phosphoglycerate
Step 9: 2-phosphoglycerate molecule loses one molecule of H2O,
producing phosphoenolpyruvate



Dehydration reaction
Enzyme: Enolase
2+
Requires Mg as cofactor (H2O molecule that is
eliminated binds to Mg2+)
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science

Step 10: phosphoenolpyruvate transfers it phosphate group to ADP,
producing ATP and pyruvate

36
quaternary structure of the tetramer can vary
according to the relative amounts of the two
kinds of subunits
Different isozymes have slightly different kinetic
properties due to their subunit composition
Five possible isozymes:
a. Skeletal Muscle - M4 (LDH 5) homogenous
tetramer; allosterically inhibited by pyruvate
b. Heart – H4 (LDH 1) homogenous tetramer; higher
affinity for lactate as substrate
c. Blood Serum – M3H, M2H2, and MH3






Enzyme: Pyruvate kinase – an allosteric enzyme consisting
for four subunits of two different types: M and L (same
with phosphofructokinase)
o Inhibited by ATP
Double bond shifts to the oxygen carbon 2 and a hydrogen
shifts to carbon 3
Phosphoenolpyruvate is the a high energy compound with
a high phosphate-group transfer potential
Free-energy of hydrolysis of this compound more negative
than that of ATP
o -61.9 kJ mol-1 vs. -30.5 kJ mol-1
o -14.8 kcal mol-1 vs. -7.3 kcal mol-1
The reaction can be considered to be the sum of the
hydrolysis of phosphoenolpyruvate
Substrate-level phosphorylation
Note: relative amounts of the H4 and MH3 isozymes in blood serum
increase drastically after myocardial infarction
Reduction of pyruvate (Waste product in aerobic organisms) the
last step in anaerobic glycolysis?
Relative amounts of NAD+ and NADH
Half reaction of reduction
Half reaction of oxidation
Overall reaction

NADH produces from NAD+ by the earlier oxidation of
glyceraldehyde-3-phosphate is used up with no net change
in the relative amounts of NADH and NAD+ in the cell
Net reaction:
ANAEROBIC METABOLISM OF PYRUVATE
Reduction of Pyruvate to Lactate
Final reaction of anaerobic glycolysis: reduction of
pyruvate to lactate

Exergonic: ∆G°' = - 25.1 kJ mol-1 = -6.0 kcal mol-1
Lactate – dead end in muscle metabolism; can be recycled in the
liver to form pyruvate and even glucose by a gluconeogenesis

Enzyme: Lactate dehydrogenase (LDH)
o An NADH-linked dehydrogenase and consist of four
subunits
o There are two types of subunits: M and H

varies slightly in amino acid compositions
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science


This regeneration is needed under anaerobic conditions in
the cell (NAD+ will be present for further glycolysis to take
place)
o Without this, oxidation reactions in anaerobic
organisms would soon come to a halt because of the
lack of NAD+ (oxidizing agent in fermentative
processes)
NADH is frequently encountered reducing agent in many
reactions; lost by organisms in lactate production
o Aerobic metabolism makes more efficient use of
reducing agents; “reducing power”
o Conversion of pyruvate to lactate does not occur in
aerobic metabolism
o NADH produced in the stages of glycolysis leading to
the production of pyruvate is available for use in other
reactions in which a reducing agent is needed

Pyruvate Conversion to Ethanol by Fermentation
Two other reactions related to the glycolytic pathway lead to the
production of ethanol by alcoholic fermentation:
1. Pyruvate is decarboxylased to produce acetaldehyde
Enzyme: Pyruvate decarboxylase



Requires Mg2+ and a thiamine pyrophosphate (TPP) –
co-factor; vitamin B1; can be found in several
decarboxylation reaction
The carbon atom between the nitrogen and the
sulfur in the thiazole ring is highly reactive - forms a
carbanion easily, and in turn, it attacks the carbonyl
group of pyruvate to form an adduct
Carbon dioxide splits off, leaving a two-carbon
fragment covalently bonded to TPP (Activated
aldehyde)
37
Acetaldehyde – formed when there is a shift of
electrons, and the two-carbon fragment splits off
o Reduced to produced ethanol; one molecule of
NADH is oxidized to NAD+ for each molecule of
ethanol produced

CO2 produced is responsible for the bubbles in beer
and in sparkling wines

Reduction reaction of alcoholic fermentation –
similar to the reduction of pyruvate to lactate
(provides for recycling of NAD+ and thus allows
further anaerobic oxidation reaction)
Net reaction:
Note: NAD+ and NADH do not appear explicitly in the net
equation. It is essential that the recycling of NADH to NAD+
takes place here

Conversion of acetaldehyde to ethanol (Alcohol
dehydrogenase) – similar to lactate dehydrogenase;
both are NADH-linked dehydrogenase, and tetramers
Cancer Detection by Lactate
Aerobic Glycolysis (Warburg effect or “Cancer’s molecular sweet
tooth”)

cancer cells exhibit a high rate of glycolysis followed by lactic
acid fermentation
takes place even when O2 is plentiful


routinely used in diagnosis of cancer by administering a
radioactively labeled analogue of glucose that binds to
hexokinase and monitoring its uptake by positron emission
tomography

Research focused on ways to modulate this effect to treat
cancer
o The number of oncogenes are activated under these
conditions
o Genes for glycolytic enzymes are activated in turn
(especially, ones that play a role in reactions to pyruvate

One possible avenue for treatment: find a suitable activator
or inhibitors of such enzymes to switch metabolism back to
the normal aerobic pathway from glycolysis
ENERGY PRODUCTION IN GLYCOLYSIS
Exergonic – overall process of glycolysis

We can calculate ∆G°' for the entire reaction by adding up
the ∆G°' values from each of the steps
Remember: all of the reactions from triose phosphate isomerase
to pyruvate kinase are doubled

The energy released in the exergonic phases pf the process
drives the endergonic reactions
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science

The net reaction of glycolysis includes an important
endergonic process, that of phosphorylation of two
molecules of ADP

Without the production ATP: the reaction of one molecule
of glucose to produce two molecules of pyruvate would be
even more exergonic (subtracting out the synthesis of ATP)
o
Energy released by the conversion of glucose to pyruvate
would be lost to the organism and dissipated as heat
o Without the production of ATP to serve as a source of
energy for other metabolic processes, the energy
released by glycolysis would serve no purpose for the
organism (except to help maintain body temperature in
warm-blooded

Energy required to produce the two molecules of ATP for
each molecule of glucose can be recovered by the organism
when the ATP is hydrolyzed in some metabolic process

The percentage of the energy released by the breakdown of
glucose to lactate that is “captured” by the organism when
ADP is phosphorylated to ATP is the efficiency of energy use
in glycolysis: (61.0/184.6) X 100 or 33%

It comes from calculating the energy used to phosphorylate
two moles of ATP as a percentage of the energy released by
the conversion of one mole of glucose to two moles of
lactate

Net release of energy in glycolysis: 123.6kJ (29.5 kcal) for
each mole of glucose converted to lactate – dissipated as
heat by the organism

Free-energy changes: are the standard values, assuming the
standard conditions (1M concentrations of all solutes except
H+

Large changes in concentrations frequently lead to relatively
small differences in the free-energy change (few kJ per mole)
Note: Concentrations under physiological conditions can differ
markedly from standard values
Some free energy changes may be different under
physiological conditions for standard conditions
CONTROL OF GLYCOLYSIS
Pathways can be “shut down” if an organism has no
immediate need for their products (saves energy for the organism)



Control is exercised near the start and end of a pathway
and at points involving key intermediates
Final step of glycolysis (major control point in glucose
metabolism – pyruvate kinase is allosterically affected by
several compounds (inhibit by ATP and alanine)
o Alanine (amino version of pyruvate); one reaction
away from pyruvate; enzyme: transaminase
o ↑ Alanine = ↑Pyruvate; enzyme that makes more
pyruvate can be shut down
o Fructose-1,6-bisphosphate allosterically activates PK
(incoming products can be processed)
Pyruvate Kinase – found as isozymes with three different
subunits:
1. M – predominates muscle
2. L – liver
3. A – other tissues
o Native pyruvate kinase molecule has four subunits –
similar to lactate dehydrogenase and
phosphofructokinase
o Liver isozymes are subject to covalent modification
Control Point (Three reactions):
1. Glucose to glucose-6-phosphate – enzyme: hexokinase
2. Production of fructose-1,6-bisphosphate – enzyme:
phosphofructokinase
3. PEP to pyruvate – enzyme: pyruvate kinase
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
38
Biochemistry for Medical Laboratory Science
o


Protein kinase phosphorylates PK (less active)l
glycolysis is shut down in the liver when blood glucose
is low
Hexokinase – inhibited by high levels of glucose-6phosphate
o When glycolysis is inhibited through
phosphofructokinase, G6P builds up, shutting down
hexokinase keeping glucose from being metabolized in
the liver
o Liver contains glucokinase (phosphorylates glucose;
has a higher Km for glucose than hexokinase; functions
only when glucose is abundant)
Example: There is an excess of glucose in the liver,
glucokinase phosphorylates it to G6P
o Purpose of this phosphorylation: it can be polymerized
into glycogen
Higher level of control is exercised by the action of
hormones
o Insulin and other hormones– control the level of
glucose in the bloodstream, activating and deactivating
metabolic pathways as needed
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
39
Biochemistry for Medical Laboratory Science
Reaction Sequence of Glycolytic Pathway
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
40
Biochemistry for Medical Laboratory Science
∆G°' and ∆G values for all the reactions of anaerobic
glycolysis and erythrocytes
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
41
Biochemistry for Medical Laboratory Science
Storage Mechanisms and Control in Carbohydrate Metabolism
HOW GLYCOGEN IS DEGRADED AND PRODUCED
42
4. Glucose-1-phosphate isomerized to give glucose-6phosphate
o Enzyme: Phosphoglucomutase
Glycogen (Animal starch) – glucose as a polymer

Found primarily in Liver and Muscles

Similar to the starches found in plants

Differs from starch in the degree of chain branching

In the degradation of glycogen, several glucose residues
can be released simultaneously, one from each end of a
branch
Glycogen: Optimized for Efficiency

Useful to organisms in meeting short-term demands for
energy by increasing the glucose supply as quickly as
possible

Mathematical Modeling shows that the structure of
glycogen is optimized for its ability to store and deliver
energy quickly and for the longest amount of time

Key to optimization: average chain length of the branches
(13 residues)
o A greater or much shorter glycogen chain would not
be as efficient as a vehicle for energy storage and
release
Glycogen Breakdown
Liver
Triggered by low levels of glucose in blood, Liver glycogen
breaks down to glucose-6-phosphate which is hydrolyzed to give
glucose

This replenishes the supply of glucose in the blood
5. Debranching reaction to hydrolyze the glycosidic bonds of
the glucose residues at branch points in the glycogen
structure
o Requires Debranching Enzymes – degrades the
α(1→6) linkages: hydrolyzes the linkages in a
branched-chain polymer such as amylopectin
o An alternative mode of entry to the glycolytic
pathway
o “Saves” one molecule of ATP for each molecule of
glucose because it bypasses the first step in glycolysis
– has a net gain of three ATP molecules for each
glycose monomer rather than two ATP molecules as
when glucose is the starting point
Note: Glycogen is a more effective energy source than
glucose but it takes energy to put the glucoses together
into glycogen
o
Debranching of glycogen involves the transfer of a
“limit branch” of three glucose residues to the end of
another branch where they are subsequently
removed by glycogen phosphorylase
o
The same glycogen debranching enzyme then
hydrolyzes the α(1→6) glycosidic bond of the last
glucose residue remaining at the branch point
Muscle
Glucose-6-phosphate obtained from glycogen breakdown
enters the glycolytic pathway directly rather than being hydrolyzed
to glucose and then exported to the bloodstream
Three reactions that play roles in the conversion of glycogen to
glucose-6-phosphate:
3. Each glucose residue cleaved from glycogen reacts with
phosphate to give glucose-1-phosphate
o Enzyme: Glycogen Phosphorylase – this enzyme
cleaves the α(1→4) linkages in glycogen
Note: this cleavage reaction is phosphorolysis rather than
hydrolysis
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
43
Glycogen breakdown is important: With low-intensity exercise, such
as jogging or long-distance running, fat is preferred, but as the
intensity increases, muscle and liver glycogen becomes more
important
Glycogen Production
Formation of glycogen from glucose is not the exact reversal of the
breakdown of glycogen to glucose
UTP – provides energy in the synthesis of glycogen; hydrolysis of a
nucleoside triphosphate
UDP-glucose or UDPG – Uridine diphosphate glucose
Addition to the First stage: a glucose residue is linked to
the tyrosine hydroxyl and glucose residues are successively
added to this first one
o Glycogenin acts as the catalyst for addition of
glucoses until there are about eight of them
linked together and then glycogen synthase takes
over
PPi – Pyrophosphate
Stage in Glycogen Synthesis
1. Glucose-1-phosphate reacts with UTP to produce UDPG
and PPi
Enzyme: UDP-glucose phosphorylase



The exchange of one phosphoric anhydride bond for
another has a free-energy close to zero
The release of energy comes about when the enzyme
inorganic pyrophosphatase catalyzes the hydrolysis of
pyrophosphate to two phosphates (Strong exergonic
reaction); Common in biochemistry
o The coupling of these two exergonic reactions to
a reaction (that is not energetically favorable)
allows an endergonic reaction to take place
UTP supply is replenished by an exchange reaction
with ATP catalyzed by nucleoside phosphate kinase:
o
2.
This makes the hydrolysis of any nucleoside
triphosphate energetically equivalent to the
hydrolysis of ATP
The addition of UDPG to a growing chain of glycogen

Each steps involves formation of a new α(1→4)
glycosidic bond in a reaction
Enzyme: glycogen synthase – must add to an existing
chain with α(1→4) glycosidic linkages; cannot form a
bond between two isolated glucose
o Because of this, a primer (hydroxyl group of a
specific tyrosine of the protein glycogenin:
37,000 Da) is required to initiate a glycogen
synthesis
3.
Glycogen synthesis requires the formation of α(1→6) and
α(1→4) glycosidic linkages
Enzyme: Branching enzyme – Catalyzes the reaction
needed to introduce a branch point during the synthesis of
glycogen
o Transferring a segment about 7 residues long
from the end of a growing chain to a branch
point where it catalyzes the formation of the
required α(1→6) glycosidic linkage
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
Note: This enzyme has already catalyzed the breaking of
an α(1→4) glycosidic linkage in the process of transferring
the oligosaccharide segment

Each transferred segment must come from a chain at
least 11 residues long; each new branch point must
be a least 4 residues away from the nearest existing
branch point
Balancing Glycogen Breakdown and Production

Simultaneous operation of both glycogen synthesis and
glycogen breakdown would result to the hydrolysis of UTP
which would also waste chemical energy stored in the
phosphoric anhydride bonds
Glycogen phosphorylase— subject to allosteric control and covalent
modification (same with the sodium-potassium pump)

This combination allows for a degree of fine tuning that
would not be possible with either mechanism alone





the enzyme is a dimer that exist in two forms:
o Inactive T (taut) – can be modified by phosphorylation of
a specific serine residue on each of the two subunits
o Active R (relaxed)
Esterification of the serines – catalyzed by phosphorylase
kinase → Phosphorylase a (Phosphorylated of glycogen
phosphorylase)
Dephosphorylation of serines – catalyzed by phosphoprotein
phosphatas → Phosphorylase b (Dephosphorylated of
glycogen phosphorylase)
switch from phosphorylase b to phosphorylase a – major
form of control over the activity of phosphorylase
Response time of the changes = seconds to minutes
Phosphorylase is also controlled more quickly in times of urgency
by allosteric effectors (milliseconds)
In liver

Glucose – allosteric inhibitor of phosphorylase a
o Binds to the substrate site and factors the transition to
the T state
o Exposes the phosphorylated serines so that the
phosphatase can hydrolyze them
o Shifts the equilibrium to phosphorylase b
44
In muscle

ATP, AMP, and glucose-6-phosphate (G6P) – allosteric
effectors
o When muscles use ATP to contract:

↑ AMP levels – stimulates formation of the R state
of phosphorylase b (active)

↑ ATP/G6P – acts as allosteric inhibitors shifting
equilibrium back to the T form
These ensures that glycogen will be degraded when there
is a need for energy
↑AMP ↓G6P↓ATP
When reverse is true, “shutting down” glycogen
phosphorylase activity is the appropriate response
Hormonal control
Epinephrine is released from the adrenal gland in response to stress,
this triggers series of events:
1. Suppresses the activity of glycogen synthase
2. Stimulate that of glycogen phosphorylase
Activity of glycogen synthase
o Same type of covalent modification as glycogen
phosphorylase but the response is opposite

Inactive form = phosphorylated form

Active = unphosphorylated
o The hormonal signals (glucagon or epinephrine)
stimulate the phosphorylation of glycogen synthase via
cAMP-dependent protein kinase (enzyme)
o Glycogen synthase is phosphorylated → inactive and
hormonal signal activates phosphorylase
o Glycogen synthase can also be phosphorylated by other
enzymes: phosphorylase kinase and enzymes called
glycogen synthase kinases
o Dephosphorylated by the same phosphoprotein
phosphatase that removes the phosphate from
phosphorylase
o Phosphorylation of glycogen synthase – more
complicated; has multiple phosphorylation sites
o As the progressive level of phosphorylation increases,
the activity of the enzyme decreases
Glycogen synthase under allosteric control
o Inhibited by ATP – can be overcome by G6P (activator)
o Two forms of glycogen synthase respond very differently
to G6P:

Glycogen synthase D – Phosphorylated (inactive) for
G6P dependent; active only under very high
concentrations of G6P (the level necessary to give
significant activity would be beyond the physiological
range)

Glycogen synthase I – nonphosphorylated form for
G6P independent; active even with low
concentrations of G6P
Even though purified enzymes can be shown to respond to
allosteric effectors, true control over the activity of
glycogen synthase is by its phosphorylation state
Two target enzymes: (1) glycogen phosphorylase; and (2) glycogen
synthase – are modified in the same way by the same enzymes links
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
45
the opposing processes of synthesis and breakdown of glycogen
more intimately
Modifying enzymes – subject to covalent modification and allosteric
control

Complicates the process considerably but adds the
possibility of an amplified response to small changes in
conditions

Small change in the concentration of an allosteric effector of
a modifying enzyme → large change in the concentration of
an active, modified target enzyme
o This amplification response is due to the fact that the
substrate for the modifying enzyme is itself an enzyme
GLUCONEOGENESIS PRODUCES GLUCOSE FROM PYRUVATE
Gluconeogenesis – the pathway of synthesis of glucose from lactate

conversion of pyruvate to glucose

Not the exact reversal of glycolysis
Pyruvate – product of glycolysis but can arise from other sources to
be the starting point of the anabolism of glucose
Essentially irreversible reaction of glycolysis are bypassed by
gluconeogenesis
Three irreversible steps in Glycolysis (the difference between
glycolysis and gluconeogenesis are found in these three reactions):
1. Production of pyruvate (and ATP) from
phosphoenolpyruvate (exergonic, reverse reaction is
endergonic)
2. Production of fructose-1,6-bisphosphate from fructose6-phosphate
3. Production of G6P from glucose

Reversing the second and third reactions would require
the production of ATP from ADP (endergonic)

The net results of gluconeogenesis includes the reversal of
these three glycolytic reactions but the pathway is
different, with different reactions and different enzymes
Pyruvate to Phosphoenolpyruvate in Two Steps
Conversion of pyruvate to phosphoenolpyruvate in gluconeogenesis
takes place in two steps:
1.
Reaction of pyruvate and carbon dioxide to give oxaloacetate



requires energy (hydrolysis of ATP)
enzyme: pyruvate carboxylase (allosteric enzyme found in
the mitochondria
Acetyl-CoA – an allosteric effector that activates pyruvate
carboxylase
o ↑Acetyl-CoA – pyruvate (precursor of acetyl-CoA) can
be diverted to gluconeogenesis
Oxaloacetate from the citric acid cycle can frequently be a
starting point for gluconeogenesis
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science


Mg2+ and biotin – also required for effective catalysis
Biotin is a carrier of CO2; has a specific site for covalent
attachment of CO2




o
2.
46
Changing the concentration of reactants or products
causes a shift to reestablish equilibrium
to the right = addition of reactants
to the left = addition of products
oxaloacetate formed in the mitochondria can have two
fates:
1. Continue to form PEP - can leave the mitochondria
via a specific transporter to continue
gluconeogenesis
2. Oxaloacetate to malate via mitochondrial malate
dehydrogenase – uses NADH; malate can then leave
the mitochondria and have the reaction reversed by
the cytosolic malate dehydrogenase
The CO2 (covalently bonded to the enzyme) is attached
to the biotin, CO2 is shifted to pyruvate to form
oxaloacetate (ATP is required)
Pyruvate carboxylase catalyzes
a compartmentalized reaction.
Pyruvate is converted to
oxaloacetate in the
mitochondria. Because
oxaloacetate cannot be
transported across the
mitochondrial membrane, it
must be reduced to malate,
transported to the cytosol, and
then oxidized back to
oxaloacetate before
gluconeogenesis can continue.
Conversion of oxaloacetate to phosphoenolpyruvate
The reason for this two-step process is that oxaloacetate cannot
leave the mitochondria, but malate can



Enzyme: phosphoenolpyruvate carboxykinase (PEPCK)
found in the mitochondria and the cytosol
This reaction also involves hydrolysis of a nucleoside
triphosphate (GTP)
Successive carboxylation and decarboxylation reactions are
both close to equilibrium (have low values of their
standard free energies)
o Result: conversion of pyruvate to
phosphoenolpyruvate is also close to equilibrium
Small ↑ oxaloacetate – can drive the equilibrium to the right
Small ↑ phosphoenolpyruvate – can drive it to the left
Law of mass action – the relationship between concentration of
products and reactants in a system at equilibrium
These two paths exist to get PEP into the cytosol because of this
enzyme:

Glyceraldehyde-3phosphate dehydrogenase – this
reaction must be reversed in gluconeogenesis, and the
cytosol has a low ratio of NADH to NAD+
The purpose pf the roundabout way of getting oxaloacetate out of
the mitochondria via malate dehydrogenase is to produce NADH in
the cytosol so that gluconeogenesis can continue
Dephosphorylation of Sugar Phosphates
The other two reactions in which gluconeogenesis differs
from glycolysis are ones in which a phosphate-ester bond to a sugar
hydroxyl group is hydrolyzed

Both reactions are catalyzed by phosphatases
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
1.

Both reactions are exergonic
Hydrolysis of fructose-1,6-bisphosphate to produce fructose-6phosphate and phosphate ion
(∆G°' = -16.7 kJ mol-1 = -4.0 kcal mol-1)
47

Two other points where the two pathways differ:
1. Conversion of fructose-6-phosphate and
fructose-1,6-bisphosphate
2. Interconversion of pyruvate and
phosphoenolpyruvate
o The control mechanisms: allosteric control and
covalent modification
o Different enzymes catalyze the opposing reactions in
the two different pathways
o figure 18.18 compares the corresponding enzymes of
the two pathways side by side, along with the
substances that activate and inhibit them

Gluconeogenesis: hydrolysis of fructose-1,6-bisphosphate
to fructose-6-phosphate
o catalyzed by fructose-1,6-bisphosphatase (subject to
allosteric inhibition by fructose-1,6-bisphosphate and
AMP)
Glycolysis: Phosphorylation of fructose-6-phosphate to
fructose-1,6-bisphosphate
o Catalyzed by phosphofructokinase (PFK)

exhibits opposite allosteric behavior activated by
fructose-1,6-bisphosphate and AMP

key enzyme of glycolysis

inhibitor of fructose bisphosphate phosphatase
(FBPase) – plays a role in gluconeogenesis

2.
Enzyme: fructose-1,6-bisphosphatase (allosteric enzyme) –
strongly inhibited by adenosine monophosphate (AMP) but
stimulated by ATP
o A control point in the pathway because of allosteric
regulation
o Ample supply of ATP; formation of glucose is favored
o Inhibited by fructose-1,6-bisphosphate (an extremely
potent activator of phosphofructokinase
Hydrolysis of glucose-6-phosphate to glucose and phosphate
ion
(∆G°' = -13.8 kJ mol-1 = -3.3 kcal mol-1)

Enzyme: glucose-6-phosphatase
In gluconeogenesis, the organism can make direct use of the
fact that the hydrolysis reactions of the sugar phosphates are
exergonic
Corresponding reactions are not the reverse of each other in the two
pathways: they differ in whether they require ATP and the enzymes
involved

Hydrolysis of glucose-6-phosphate to glucose occurs in the
endoplasmic reticulum
o An example of an interesting pathway that requires
three cellular locations (mitochondria, cytosol, and
endoplasmic reticulum)
CONTROL OF CARBOHYDRATE METABOLISM
Reciprocal Regulation in Glucose Metabolism
Reciprocally regulated: Glycolysis and gluconeogenesis,
and the breakdown and synthesis of glycogen
Glycolysis and Gluconeogenesis

Glucose to G6P in Glycolysis differs from, the reverse
reaction in gluconeogenesis
o Different enzymes
o Key feature - Binding of G6P to the phosphatase with
reaction velocity primarily depended on the substrate
concentration

“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
48
o


The intracellular level of fructose-1,6-bisphosphate
(F2,6BP) is a key point in the structure above
o F2,6BP is an important allosteric activator for PFK
o High concentration of F2,6BP stimulates glycolysis
o Low concentration of F2,6BP stimulate
gluconeogenesis
o Concentration depends in the balance between its
synthesis (phosphofructokinase-2 or PFK-2) and its
breakdown (fructose-1,6-bisphosphatase-2 or FBPase2)
o PFK-2 and FBPase-2 are controlled by
phosphorylation/dephosphorylation mechanisms

The figure shows that the inhibitor works by itself, but
its effect is greatly increased by the presence of the
allosteric inhibitor (AMP)
The breakdown and synthesis of glycogen exhibits similar
reciprocal regulation
o Covalent modification and allosteric control in
glycogen synthase and glycogen phosphorylase
o Hormonal control is of paramount importance in
glycogen synthesis and breakdown
Enzymatic cascade triggers the activation of glycogen
phosphorylase
o
o
o
The figure shows the chain of events the ultimately
leads to the activation of glycogen phosphorylase
o
Shows the first reaction in this cascade – adenylate
cyclase reaction

Product: (a) cyclic AMP—activator of the next
kinase in the series); (b) Pyrophosphate—
hydrolysis of this releases energy to drive the
reaction
o End product: Active phosphorylated form of glycogen
phosphorylase a
Phosphorylation of the dimeric protein leads to an
increase in activity of FBPase-2 and a decrease in the
concentration of F2,6BP (stimulating
gluconeogenesis)
Dephosphorylation of the dimeric protein leads to an
increase in PFK-2 activity and an increase in the
concentration of F2,6BP (stimulating glycolysis)
Note: Covalent modification is a more important feature
than allosteric interaction in regulating glycogen breakdown

Glycogen synthesis: an enzymatic cascade triggered by
insulin binding to receptors on the cell surface starts series
of covalent modification (activates glycogen synthase
kinase)
Note: Various aspects of glucose metabolism are connected
by the many levels of reciprocal regulation
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
49
Hormone Control of Glycogen Metabolism
Three important hormones in the regulation of carbohydrate
metabolism:
1. Insulin— secreted by the β-cells of the islets of Langerhans
in the pancreas (increased blood glucose levels)

Uptake of insulin by cells triggers the protein kinase
cascade that eventually leads to glycogen synthesis]

Another protein kinase cascade stimulates the action
of GLUT4 (glucose transport protein)
o GLUT 4 is released from vesicles within the cell to
the cell surface bringing glucose into the cell
o Glucose is then converted to G6P which have to
fates:
a. Incorporated into glycogen
b. Serve as an allosteric activator of the
phosphorylated glycogen synthase
General Methods to Regulate Metabolism
Important Mechanisms of Metabolic Control: applies to all aspects of
metabolism
These two hormones plays an important role when blood glucose
levels decrease:
2. Glucagon— is a peptide hormone secreted by the cells of
the islets of Langerhans; operates with insulin over a
longer time scale to stabilize blood glucose levels
3. Epinephrine (adrenaline)— an amino acid derivative;
important in the very short time scale of the “fight or
flight” response
Binding of either these hormones initiates a cascade that activates
glycogen phosphorylase and inhibits glycogen synthase
Note: Response time of a given control mechanism can be one of its
most important features
Substrate Cycling refers to the fact that opposing reactions can be
catalyzed by different enzymes

Consequently, the opposing reactions can be
independently regulated and have different rates
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
Note: different rate with the same enzyme is not possible because a
catalyst speeds up a reaction and the reverse of the reaction to the
same extent

Example: Conversion of fructose-6-phosphate to fructose-1,6bisphosphate and then back to of fructose-6-phosphate

Reaction catalyzed by phosphofructokinase (highly
exergonic)
∆G = -25.9kJ mol-1 = -6.2 kcal mol-1


Opposing reaction (part of gluconeogenesis), catalyzed by
fructose-1,6-bisphosphatase (exergonic)
∆G = -8.6kJ mol-1 = -2.1 kcal mol-1
Note: opposing reactions are not the exact reverse of one another.
Two opposing reactions + net reaction

Hydrolysis of ATP is the energetic price that is paid for
independent control of opposing reactions
Glycogen Metabolism in Body Organs



50
Gluconeogenesis recycles the lactate that is produces
o The process occurs to a great extent in the liver after
the lactate is transported there by the blood (becomes
and energy store for the next burst of exercise)
There is a division of labor between wo different types of
organs: (1) Muscle; and (2) Liver
Glycolysis and Gluconeogenesis are not highly active
simultaneously
o When the cell needs ATP = glycolysis is more active
o Little need for ATP = gluconeogenesis is more active
Hydrolysis of ATP and GTP causes the reactions of
gluconeogenesis, the overall pathway, as exergonic
∆G = -37.6kJ mol-1 = -9.0 kcal mol-1
Conversion of pyruvate to lactate is exergonic; the reverse
reaction is endergonic
o Energy released by the exergonic conversion of
pyruvate to glucose by gluconeogenesis facilitates the
endergonic conversion of lactate to pyruvate
Note: Cori cycle required the net hydrolysis of two ATP and two GTP
ATP is produced by the glycolytic part of the cycle, but the
portions involving gluconeogenesis requires yet more ATP in
addition to GTP
An organism can set up a division of labor among tissues
and organs to maintain control of glucose metabolism using
combinations of these control mechanisms (ex. cori cycle)
Hydrolysis of both ATP and GTP is the price of increased
simultaneous control of the two opposing pathways
GLUCOSE IS SOMETIMES DIVERTED THROUGH THE PENTOSE
PHOSPHATE PATHWAY
Cori Cycle named for Gerty and Carl Cori (first people who described
it)

There is a cycling of glucose due to glycolysis in muscle and
gluconeogenesis in liver

Glycolysis in fast-twitch skeletal muscle has produces
lactate (under conditions of oxygen debt)

Fast-twitch muscle has few mitochondria- metabolism is
largely anaerobic

Build-up of lactate contributes to the muscular aches that
follow strenuous exercise
Pentose Phosphate Pathway a pathway in sugar metabolism that
give rise to five-carbon sugars and NADPH

An alternative to glycolysis and differs from it in several
important ways

Production of ATP is not the crux of the matter

Five-carbon sugars are produced from glucose (ex. ribose)

PPP begins with a series of oxidation reactions that
produce NADPH and five-carbon sugars

Remainder of the pathway involves nonoxidative
reshuffling of the carbon skeletons of the sugars involved
o Products of this reaction: fructose-6-phosphate and
glyceraldehyde-3-phosphate

Important: production of Nicotinamide Adenine
Dinucleotide Phosphate (NADHP)
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
2.
51
Ribulose-5-phosphate isomerizes to ribose-5-phosphate
Enzyme: phosphopentose isomerase
Production of sugar with an aldehyde group rather
than a ketone

Ribose-5-phosphate is necessary building block for the
synthesis of nucleic acid and coenzymes (NADH)



Comparison of NADH and NADHP
o NADHP – contains one extra phosphate group
esterified to the ribose ring of the adenine nucleotide

A reducing agent in biosynthesis
o NADH— Produced in the oxidative reactions that give
rise to ATP
Pentose Phosphate Pathway: Oxidative
Oxidative reactions of the Pentose Phosphate Pathway:
1. G6P is oxidized to 6-phosphogluconate producing NADPH

Enzyme: glucose-6-phosphate dehydrogenase
2. 6-phosphogluconate molecule loses its carboxyl group
which is released as CO2

Enzyme: 6-phosphogluconate dehydrogenase

An oxidative decarboxylation reaction produces
NADPH

C-3 hydroxyl group of the 6-phospphogluconate is
oxidized to form a β-keto acid (unstable and readily
decarboxylates to form ribulose-5-phosphate
Pentose Phosphate Pathway: Nonoxidative
Two different reactions in which ribulose-5-phosphate isomerizes:
1. Inversion of configuration around carbon atom 3
producing xylulose-5-phosphate

Enzyme: phosphopentose-3-epimerase
Group-transfer reactions that link the PPP with glycolysis
require the two five-carbon sugars produced by the isomerization of
ribulose-5-phosphate

Two molecules of xylulose-5-phosphare and one
molecule of ribose-5-phosphate rearrange to give two
molecules of fructose-6-phosphate (hexose) and one
molecule of glyceraldehyde-3-phosphate (triose)

The total number of carbon atoms (15) does not
change, but there is considerable rearrangement as a
result of group transfer
Two enzymes responsible for the reshuffling of the carbon atoms of
sugars (R5P; X5P) in the remainder of the pathway:
1. Transketolase
2. Transaldolase
In three reactions:

Transketolase transfer a two-carbon unit from
xylulose-5-phosphate to ribose-5-phosphate to give
sedoheptulose-7-phosphate
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science

Transaldolase trasfers a three-carbon unit from the
sedoheptulose-7-phosphate to glyceraldehyde-3phosphate
52
Note: Control mechanisms of the PPP can respond to the varying
needs of organisms for either or both of these compounds
Pentose Phosphate Pathway: Regulation

Transketolase catalyzes the reaction between
xylulose-5-phosphate and eryhtrose-4-phosphate
producing fructose-6-phosphate and glyceraldehyde3-phosphate
Reactions catalyzed by transketolase and transaldolase are
reversible allowing PPP to respond to the needs of an organism

Plays an important role in the organism’s ability to
adjust its metabolism to changes in conditions
o Transaldolase— both an aldol cleavage and an
aldol condensation occur at differen stages of the
reaction
o Transketolase— resembles pyruvate
decarboxylase; requires Mg2+ and thiamine
pyrophosphate (TPP)

G6P undergoes different reactions (depending on
whether there is a greater need for ribose-5-phosphate
or for NADPH

Operation of the oxidative portion depends strongly on
the organisms requirement for NADPH (the need for
ribose-5-phosphater can be met in other ways;
obtained from glycolytic intermediates)

If NADPH is more needed then ribose-5-phosphate,
reaction series go through the complete pathway

Oxidative reactions are needed to produce NADPH.
Net reaction:

If ribose-5-phosphate is needed more than NADPH,
fructose-6-phosphate and glyceraldehyde-3-phosphate
can give rise to ribose-5-phosphate (bypassing the
oxidative portion of the PPP)
Results:
In the PPP, G6P can be converted to fructose 6 phosphate
and glyceraldeyde-3-phosphate other than the glycolytic pathway
hence it is also called hexose monophosphate shunt
Major feature: production of ribose-5-phosphate and NADPH
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
o
CITRIC ACID CYCLE
THE CENTRAL ROLE OF THE CITRIC ACID CYCLE IN METABOLISM
Organism can obtain far more energy from nutrients by aerobic
oxidation than by anaerobic oxidation

30 to 32 molecules of ATP can be produced from each
molecule of glucose in complete aerobic oxidation to CO2
and H2O
Three processes play roles in aerobic metabolism: operates
together in aerobic metabolism
1. Citric Acid Cycle
2. Electron transport
3. Oxidative Phosphorylation

Metabolism consist of:
o Catabolism – oxidative breakdown of nutrients
o Anabolism – reductive synthesis of biomolecules

Citric Acid Cycle
o aka Tricarboxylic Pathway – some of the molecules
involved are acids with three carboxyl groups
o aka Kreb’s Cycle – named after Sir Hans Krebs
o Amphibolic – plays a role in both catabolism and
anabolism
o Some molecule included in this cycle are the starting
points of biosynthetic (anabolic) pathways
Note: Metabolic pathways operate simultaneously
THE OVERALL PATHWAY OF THE CITRIC ACID CYCLE
Important Difference between Glycolysis and CAC (Eukaryotes):
Glycolysis – occurs in the cytosol
CAC – occurs in the mitochondria
Mitochondrial Structure
Mitochondrion – has an inner (tight barrier between the matrix and
the cytosol; very few compounds can cross this barrier) and outer
membrane
53
Enzymes that catalyzed FAD-linked reaction is an integral
part of the inner mitochondrial membrane and is linked
directly to the electron transport chain
Overview of Reactions in the Citric Acid Cycle
Schematic form of the Citric Acid Cycle
Under aerobic conditions, pyruvate produced by glycolysis is
oxidized further with CO2 and H2O as the final products
1. The pyruvate is oxidized to one CO2 molecule and to one
acetyl group (becomes linked to an intermediate,
coenzyme A (CoA)
2. The acetyl-CoA enters the CAC
3. In the CAC, two more molecules of carbon dioxide are
produced for each molecules of acetyl-CoA that enters the
cycle, and electrons are transferred in the process
4. The immediate electron acceptor in all cases but one is
NAD+, which is reduced to NADH
5. In the one case, Flavin adenine dinucleotide (FAD), which is
derived from riboflavin (vitamin B2), takes up two
electrons and two hydrogen ions to produce FADH2
6. The electrons are passed from NADH and FADH2 through
several stages of an electron transport chain with a
different redox reaction at each step
7. The final electron acceptors is oxygen, with water as the
product
Note: starting from pyruvate, a three-carbon compound, three
carbons are lost as CO2 via the production of acetyl-CoA and one
turn of the cycle
8. The cycle produced energy in the form of reduced electron
equivalents, but the carbon skeletons are effectively lost
Note: the cycle also produced one high-energy compound directly,
guanosine triphosphate (GTP)




In the first reaction: two-carbon acetyl group condenses with
the four-carbon oxaloacetate ion to produce the six-carbon
citrate ion
Next few steps: Oxidative decarboxylation— citrate
isomerizes, both loses CO2 and is oxidized; produces the fivecarbon compound α-ketoglutarate (oxidatively
decarboxylated) to produce he four-carbon compound
succinate
The cycle is completed by regeneration of oxaloacetate form
succinate in several steps
CAC has eight steps:
o Steps 3, 4,6, and 8 – oxidation reactions; oxidizing agents
is NAD+ except step 6 (FAD)
o Step 5 – guanosine diphosphate (GDP) phosphorylated
to produce GTP; equivalent to the production of ATP
HOW PYRUVATE IS CONVERTED TO ACETYL-COA



Mitochondrial matrix – region enclosed by the inner
membrane
Intermembrane space – exist between the inner and outer
membranes
The reactions of CAC take place in the matrix except for the
one in which the intermediate electron acceptor is FAD
Pyruvate dehydrogenase – enzyme responsible for the conversion of
pyruvate to CO2 and the acetyl portion of acetyl-CoA

There is an –SH group at one end of the CoA molecule (the
point at which the acetyl group is attached

CoA is frequently shown in equations as CoA-SH as a result—
CoA is a thiol, acetyl-CoA is a thioester, with a sulfur atom
replacing an oxygen of the usual carboxylic ester
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
Note: this difference is important since thioesters are high-energy
compounds (hydrolysis of thioesters releases enough energy to
drive other reactions)

Oxidation reaction precedes the transfer of the acetyl group
to the CoA

Overall Reaction: Exergonic
Pyruvate to acetyl-CoA
Five enzymes that make up the pyruvate dehydrogenase complex
in mammals:
1. Pyruvate dehydrogenase (PDH)

Catalyzes pyruvate to CO2 and Acetyl-CoA

Requires: Thiamine pyrophosphate (TTP) – coenzyme
and Mg2+

Not covalently bonded to the enzyme; they are held
together by noncovalent interactions

α-keto acid, pyruvate, loses CO2; the remaining twocarbon unit becomes covalently bonded to TPP
2. Dihydrolipoyl transacetylase
o Requires: Lipoic acid – coenzyme (Covalently bonded
to the enzyme by an amide bond to the ɛ-amino group
of lysine side chain)

The two-carbon unit that came from pyruvate is
transferred from the thiamin pyrophosphate to the
lipoic acid; a hydroxyl group is oxidized to produce an
acetyl group

Oxidizing agent: disulfide group of the lipoic acid
(reduced)

Product of reaction: Thioester
3. Dihydrolipoyl dehydrogenase

A molecule of CoA-SH attacks the thioester linkage,
and the acetyl group is transferred to it

The acetyl group remains bound in a thioester linkage;
this time it appears as acetyl-CoA

The reduced form of lipoic acid remains covalently
bound to Dihydrolipoyl transacetylase
54

The reaction of pyruvate and CoA-SH reached the
stage of products (CO2 and acetyl-CoA)

The rest of the steps regenerate the lipoic acid (further
reactions can be catalyzed by the transacetylase)
4. Pyruvate dehydrogenase kinase – flavoprotein (their
attachment to FADs)

Reoxidized th4e reduced lipoic acid from the sulfhydryl
to the disulfide form

Lipoic acid still remains covalently bonded to the
transacetylase enzyme

Coenzyme: FAD - bound to enzyme by noncovalent
interactions; reduced to FADH2 (reoxidized in turn)

Oxidizing agent: NAD+

Product: NADH along with reoxidized FAD

Reduction of NAD+ to NADH accompanies the oxidation
of pyruvate to the acetyl group; there has been
transfer of two electrons from pyruvate to NAD+

Electrons gained by NAD+ is generating NADH in this
step are passed to the electron transport chain

Reaction leading from pyruvate to acetyl-CoA is a
complex one
o Requires three enzyme (has its own coenzyme in
addition to NAD+)
o Spatial orientation of the individual enzyme
molecules with respect to one another is complex
o This makes compact arrangement possible – in
which the various stages of the reaction can
proceed very efficiently
5. Pyruvate dehydrogenase phosphatase
Involved in the conversion of pyruvate to acetyl-CoA


Kinase and Phosphatase are enzymes used in the control
of PDH and are present on a single polypeptide
o The reaction takes place in five steps
o Two enzymes catalyze reactions of lipoic acid (has
disulfide group in its oxidized form), and two sulfhydryl
groups in its reduced form
Lipoic acid – act as an oxidizing agent or can
simultaneously take part in two reactions (REDOX and the
shift of an acetyl group by transesterification
o Reaction involves hydrogen transfer (frequently
accompanies biological oxidation-reduction reactions
o Formation of thioester linkage with the acetyl group
before it is transferred to the acetyl-CoA
Compact arrangement has two advantages
1. Various stages of the reaction can take place more
efficiently because the reactants and the enzymes are
so close to each other
o The lipoic acid and the lysine side chain to which
it I bonded are long enough to act as “swinging
arm” – can move to the site of each of the steps
of the reaction; the lipoic acid can move to the
pyruvate dehydrogenase site to accept the twocarbon unit and then transfer it to the active site
of the transacetylase
o The acetyl group can be transesterified to CoASH from the lipoic acid
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
2.
o The lipoic acid can swing to the active site of the
dehydrogenase so that the sulfhydryl groups can
be reoxidized to a disulfide
Regulatory controls can be applied more efficiently in
such a system than in a single enzyme molecule
o Controlling factors are intimately associated with
the multi-enzyme complex itself
55
Step 3. Formation of α-Ketoglutarate and CO2—First Oxidation
THE INDIVIDUAL REACTIONS OF THE CITRIC ACID CYCLE
Step 1. Formation of Citrate








Reaction of acetyl-CoA and oxaloacetate to form citrate and
CoA-SH – Condensation (New carbon is formed)
This reaction is followed by the hydrolysis of the citryl-CoA to
give citrate and CoA-SH
Enzyme: citrate synthase (condensing enzyme)
Synthase – an enzyme that makes new covalent bond during
the reaction; does not require the direct input of ATP
(exergonic reaction—hydrolysis of thioester releases energy)
Oxidative decarboxylation of isocitrate to α-ketoglutarate
and CO2
Enzyme: isocitrate dehydrogenase
Two steps: (1) isocitrate is oxidized to oxalosuccinate –
remains bound to the enzyme; (2) oxalosuccinate is
decarboxylates and the CO2 and α-ketoglutarate are
released
First reaction in which NADH is produced – one molecule
of NADH is produced from NAD+ at this stage by the loss of
two electrons in the oxidation
Step 4. Formation of Succinyl-CoA and CO2—Second Oxidation
Note: Thioesters are considered high-energy compounds
Step 2. Isomerization of Citrate to Isocitrate







Enzyme: Aconitase –requires Fe2+; can select one end of the
citrate molecule in preference to the other
Citrate (achiral) is converted to isocitrate (chiral)
It is often possible for a chiral compound to have several
different isomers – isocitrate has four possible isomers; this
means that the enzyme can bind a symmetrical substrate in
an unsymmetrical binding site
Intermediate Cis-asconitate – remains bound to the enzyme
during the course of the reaction
o Citrate is complexed to the Fe(II) in the active site of the
enzyme in such a way that the citrate curls back on itself
in a nearly circular conformation (ferrous wheel)







Succinyl-CoA and CO2 are formed from α-ketoglutarate and
CoA
The reaction occurs in several stages and is catalyzed by an
enzyme system (α-ketoglutarate dehydrogenase complex)
Each of these multienzyme systems consists of three
enzymes that catalyze the overall reaction
Requires: Thiamine pyrophosphate (TPP), FAD, lipoic acid,
and Mg2+
Enzyme: pyruvate dehydrogenase
Exergonic reaction
Two molecules of CO2 have been produced by the
oxidative decarboxylation of the citric acid cycle
Removal of the CO2 makes the citric acid cycle irreversible
in vivo (in vitro – separate reaction is reversible)
Two molecules of CO2 arise from carbon atoms that were
aprt of the oxaloacetate with which the acetyl group
condesned
The carbons of this acetyl group are incorporated into the
oxaloacetate (regenerated for the next round)
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
56
Step 5. Formation of Succinate






The thioester bond of succinyl-CoA is hydrolyzed to produce
succinate and CoA-SH; accompanying reaction:
phosphorylation of GDP to GTP
Enzyme: succinyl-CoA synthetase
Synthetase – enzyme that create a new covalent bond and
requires the direct input of energy from a high-energy
phosphate


Succinate is oxidized to fumarate
Enzyme: succinate dehydrogenase (falvoprotein - Flavin
moiety) – integral protein of the inner mitochondrial
membrane
o Contains iron atoms but does not contain a heme
group (nonheme iron protein; iron-sulfur protein—
protein contains several cluster that consist of four
atoms each of iron and of sulfur)
FAD is covalently bonded to the enzyme; FAD is reduced to
FADH2 and succinate is oxidized to fumarate
Overall reaction:
E-FAD and E-FADH2 – indicates that the electron acceptor
is covalently bonded to the enzyme
o E-FADH2 group passes electrons on to the electron
transport chain → oxygen = 1.5ATP
Step 7. Formation of L-Malate
Note: difference between: synthase – does NOT require energy
from phosphate-bond hydrolysis; and synthetase – requires energy
from phosphate-bond hydrolysis



Phosphate group covalently bonded to the enzyme is directly
transferred to the GDP
o The phosphorylation of GDP to GTP (Endergonic)
Energy required for the phosphorylation of GDP to GTP is
provided by the hydrolysis of succinyl-CoA to produce
succinate and CoA
Overall reaction: slightly exergonic – as a result, does not
contribute greatly to the overall production of energy by the
mitochnodira
Note: the name of the enzyme describes the reverse reaction

Enzyme that catalyzes the transfer of phosphate group
from GTP to ADP to give GDP and ATP:
nucleosidediphosphate kinase (substrate-level
phosphorylation) – to distinguish it from the type of reaction
for production of ATP that is coupled to the electron
transport chain

The distinction between substrate-level phosphorylation and
oxidative phosphorylation is important
o Substrate-level phosphorylation (free energy hydrolysis
of succinyl-CoA) provides the energy for the
phosphorylation reaction
o Production of ATP in this reaction is the only place in the
citric acid cycle in which chemical energy (ATP) is made
available to the cell
Steps 6 to 8 – four carbon succinate ion is converted to oxaloacetate
ion is converted oxaloacetate ion to complete the cycle
Step 6. Formation of Fumarate—FAD-linked Oxidation



Enzyme: fumarase
Water is added across the double bond of fumarate in a
hydration reaction to give malate
There is sterospecificity in the reaction
Step 8. Regeneration of Oxaloacetate—Final Oxidation

Malate is oxidized to oxaloacetate; another molecule of
NAD+ is reduced to NADH

Enzyme: malate dehydrogenase

Oxaloacetate can then react with another molecule of
acetyl-CoA to start another round of the cycle

Oxidation of pyruvate by pyruvate dehydrogenase
complex, and CAC → production of three CO2 molecules

One molecule of GDP is phosphorylated to GTP, one
molecule of FAD is reduced to FADH2, and four molecules
of NAD+ are reduced to NADH (3 from CAC, 1 from reaction
of pyruvate dehydrogenase complex)

Overall stoichiometry: sum of pyruvate dehydrogenase
reaction and the CAC cycle
Note: only one high-energy phosphate, GTP, is produced directly
from the CAC, but many more ATP will arise from the reoxidation of
NADH and FADH2
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
57
Control of the CAC is exercised at three points

Two ATP produced per glucose in glycolysis and two NADH
(give rise to another five ATP)
Note: Most important reaction— have important cofactors (NADH,
FADH2, GTP); Steps where CO2 is given off
These reaction play a large role in the cycle’s contribution
to our metabolism

The three decarboxylations mean that for every three
carbons entering pyruvate, three carbons are effectively
lost during the cycle
ENERGETICS AND CONTROL OF THE CITRIC ACID CYCLE
Energetics of the Citric Acid Cycle


Pyruvate to acetyl-CoA – Exergonic
CAC – Exergonic
Pre-cycle Regulation





The enzyme that initiates is is inhibited by ATP and NADH –
both compounds are abundant when a cell has a good deal
of energy readily available
The end products of a series of reaction inhibit the first
reaction of the series
The intermediate reactions do not take place when their
products are not needed
Pyruvate dehydrogenase (PDH) complex is activated by
ADP (abundant when cell needs energy)
Phosphate group – covalently bound to the enzyme in a
reaction catalyzed by pyruvate dehydrogenase kinase
o When the need arises for it to be activated, hydrolysis
of the phosphate ester linkage is catalyzed by another
enzyme (phosphoprotein phosphatase – activated by
Ca2+)
Note: Both enzymes are associated with the mammalian pyruvate
dehydrogenase complex, permitting effective control of the overall
reaction from pyruvate to acetyl-CoA



Individual reactions of the cycle, only one is strongly
endergonic: oxidation of malate to oxaloacetate
However, coupled to one of the strongly exergonic
reactions of the cycle, the condensation of acetyl-CoA and
the oxaloacetate to produce citrate and coenzyme A
There is more release of energy to come in the electron
transport chain
o When the four NADH and single FADH2 produced by
the pyruvate dehydrogenase complex and CAC are
reoxidized by the electron transport chain,
considerable quantities of ATP are produced


PDH kinase and PDH phosphatase: found in the same
polypeptide chain
o High levels of ATP activate the kinase
Pyruvate dehydrogenase is inhibited acetyl-CoA for CAC
comes from other sources
Regulatory Enzymes that Control the Citric Acid Cycle
Three control points are the reactions catalyzed by:
1. Citrate Synthase – allosteric enzyme inhibited by ATP, NADJ,
succinyl-CoA, and citrate
2. Isocitrate Dehydrogenase – ADP and NAD+ are allosteric
activators of the enzyme

ATP and NADH inhibit enzymes of the pathway, and
ADP and NAD+ activate these enzymes
3. α-ketoglutarate Dehydrogenase Complex – ATP and NADH
are inhibitors and Succinyl-CoA
Note: when a cell is metabolically active, it uses ATO and NADH at a
great rate, producing large amounts of ADP and NAD+
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science




58
low NADH/NAD+ ratio is also characteristic of an active
metabolic state
A resting cell has fairly high levels of ATP and NADH
The ATP/ADP ratio and the NADH/NAD+ ratio are also high
in resting cells (which do not need to maintain a high level
of oxidation to produce energy)
Cells with low energy requirement (high “energy charge”):
High ATP/ADP and NADH/NAD+ ratios, the presence of so
much ATP and NADH serves as a signal to “shut down” the
enzymes responsible for oxidative reactions
o Low “Energy charge”: this is characterized by low
ATP/ADP and NADH/NAD+ ratios; the need to release
more energy and to generate more ATP serves as a
signal to “turn on” the oxidative enzymes
Note: this relationship of energy requirements to enzyme activity is
the bases for overall regulatory mechanism exerted at a few control
points in metabolic pathways
THE CITRIC ACID CYCLE IN CATABOLISM
First step in the breakdown of nutrients:
Degradation of large molecules to smaller ones

Polysaccharides are hydrolyzed by specific enzymes to
produce sugar monomers
Example: breakdown of starch by amylases; Lipases hydrolyze
triacylglycerols to give fatty acids and glycerol; Protein digested by
proteases (amino acids as end product)
Figure 19.23: Various catabolic pathways that feed into the citric
acid cycle; Outline of pathways by which amino acids are converted
to components of the CAC

Catabolic reactions occur in the cytosol, CAC takes place in
the mitochondria

Many of the end products of catabolism cross the
mitochondrial membrane and then participate in the CAC
Note: All pathways leads to the CAC
Note: The supply of oxaloacetate would soon be depleted if there
were no means of producing it from a readily available source
The Citric Acid Cycle in Lipid Anabolism
Starting point of Lipid Anabolism: acetyl-CoA (both plants&animals)

Anabolic reactions of lipid metabolism take place in the
cytosol

These reactions are catalyzed by soluble enzymes that are
not bound to membranes

Acetyl-CoA – mainly produced by the mitochondria

Indirect transfer mechanisms exists for transfer of acetylCoA in which citrate is transferred to the cytosol
THE CITRIC ACID CYCLE IN ANABOLISM
CAC is a source of starting materials for the biosynthesis of
many important biomolecules, but the supply of the starting
materials that are components of the cycle must be replenished if
the cycle is to continue operating.

Oxaloacetate in an organism must be maintained at a level
sufficient to allow acetyl-CoA to enter the cycle

Anaplerotic reaction – reaction that replenishes CAC
immediately

In some organisms, acetyl-CoA can be converted to
oxaloacetate and other CAC intermediates by the
glyoxylate cycle except mammals
o In mammals, oxaloacetate is produces from pyruvate
by the enzyme pyruvate carboxylase
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
59
Figure 19.32. Overall outline of anabolic reactions



Citrate reacts with CoA-SH to produce citryl-CoA (cleaved
to yield oxaloacetate and acetyl-CoA)
The enzyme that catalyzes this reaction requires ATP: ATPcitrate lyase
Overall reaction:

Oxaloacetate can be reduced to malate



The similarity of the two schematic diagrams points out
that catabolism and anabolism (closely related)
The operation of any metabolic pathways, can be
“speeded up” or “slowed down” in response to the needs
of an organisms by control mechanisms (feedback control)

Malate can move into and out of the mitochondria by
active transport processes
o Malate produced in this reaction can be sued again in
the CAC, however, malate need no to be transported
back into the mitochondria (but can be oxidatively
decarboxylated to pyruvate by malic enzyme – requires
NADP+)
Note: these two reaction are a reduction reaction followed by an
oxidation; no net oxidation, but there is a substitution of NADPH for
NADH (Many of the enzymes of fatty acid synthesis require NADPH)
Pentose Phosphate pathways is also a source of NADPH
(principle source)

The two ways or producing NADPH indicates that all
metabolic pathways are related
Amino Acids and the Citric Acid Cycle
Starting point of the anabolic reaction that produces amino acids:
Intermediates of the CAC that can cross the mitochondrial
membrane into the cytosol

Oxaloacetate can undergo a transamination reaction to
produce aspartate, and aspartate can undergo further
reactions to form not only amino acids but also other
nitrogen-containing metabolites (pyrimidines)

Glutamate arises from α-ketoglutarate as a result of
another transamination reaction
o Glutamate undergoes further reactions to form still
more amino acids

Succinyl-CoA gives rise not to amino acids buyt to the
porphyrin ring of the heme group
o First reaction of the heme biosynthesis (condensation
of succinyl-CoA and glycine to form δ-aminoleyunilic
acis (takes place in the mitochondrial matrix; the
remainder of the pathway occurs in the cytosol)
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
60
Biochemistry for Medical Laboratory Science
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
61
Biochemistry for Medical Laboratory Science
62
NUCLEOTIDES, NUCLEIC ACIDS, AND GENETIC INFORMATION
Nucleotides – participate in oxidation–reduction reactions, energy
transfer, intracellular signaling, and biosynthetic reactions.
Polymers: Nucleic acids (DNA and RNA)
Nucleic acid – primary players in the storage and decoding of genetic
information.
perform structural and catalytic roles in cells.
NUCLEOTIDES
ubiquitous molecules with considerable structural diversity.
with eight common varieties, each composed of a
nitrogenous base linked to a sugar to which at least one
phosphate group is also attached.
planar, aromatic, heterocyclic molecules that are structural
derivatives of either purine or pyrimidine.
Major purines: Adenine (A) and Guanine (G)
form bonds to a five-carbon sugar (a pentose) via their N9
atoms
Major pyrimidines: Cytosine (C), Uracil (U), and Thymine (T)
form bonds to a five-carbon sugar through their N1 atoms
Ribonucleotides – pentose is ribose
Deoxyribonucleotides – sugar is 2’-deoxyribose
-
diffuses throughout the cell to provide energy for other
cellular work, such as biosynthetic reactions, ion transport,
and cell movement.
its chemical potential energy is made available when it
transfers one (or two) of its phosphate groups to another
molecule – hydrolysis of ATP to ADP.
NUCLEIC ACID STRUCTURE
Nucleic acid – chains of nucleotides whose phosphates bridge the 3′
and 5′ positions of neighboring ribose units.
acidic and polyanions
Phosphodiester bond – linkage between individual nucleotides.
Nucleotide residue – nucleotide that has been incorporated into the
polynucleotide.
a. 5’ end – terminal residue whose C5′ is not linked to another
nucleotide.
b. 3’ end – terminal residue whose C3′ is not linked to another
nucleotide
Nucleoside – phosphate group is absent
Adenosine triphosphate (ATP) – a nucleotide containing adenine,
ribose, and a triphosphate group.
an energy carrier or energy transfer agent.
formed from adenosine diphosphate (ADP) through the
process of photosynthesis or the breakdown of metabolic
fuels such as carbohydrates and fatty acids.
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
Chargaff’s rules – discovered in the late 1940s by Erwin Chargaff, who
devised the first reliable quantitative methods for the compositional
analysis of DNA.

DNA has equal numbers of adenine and thymine residues
(A = T) and equal numbers of guanine and cytosine residues
(G = C)
DNA Forms a Double Helix
Watson-Crick structure – by James Watson and Francis Crick in 1953.
determination of the structure of DNA
molecular mechanism of heredity
Tautomers – readily interconverted isomers that differ only in
hydrogen positions.
DNA’s helical structure – provided by an X-ray diffraction photograph
of a DNA fiber taken by Rosalind Franklin.
a. DNA is a helical molecule
b. its planar aromatic bases form a stack that is parallel to the
fiber axis.
B-DNA – most common form of the DNA double helix.
A-DNA – a form of a DNA double helix characterized by having fewer
residues per turn and major and minor grooves with dimensions that
are more similar to each other than those of B-DNA.
Z-DNA – a form of DNA that is a left-handed helix, which has been
seen to occur naturally under certain circumstances.
63
vice versa, and each guanine residue must pair with a
cytosine residue and vice versa (Fig. 3-8). These hydrogenbonding interactions, a phenomenon known as
complementary base pairing, result in the specific
association of the two chains of the double helix.
Each DNA strand can act as a template for the synthesis of its
complementary strand and hence that hereditary information is
encoded in the sequence of bases on either strand.
Supercoils – extra twists (over and above those of the double helix) in
closed circular DNA.
Negative supercoils – circular DNA with fewer than the normal
number of turns of the helix.
Topoisomerases – enzymes that relax supercoiling in closed circular
DNA.
The Watson–Crick model of DNA has the following major features:
Two polynucleotide chains wind around a common axis to form a
double helix.
1. The two strands of DNA are antiparallel (run in opposite
directions), but each forms a right-handed helix.
2. The bases occupy the core of the helix and sugar–phosphate
chains run along the periphery, thereby minimizing the
repulsions between charged phosphate groups. The surface
of the double helix contains two grooves of unequal width:
the major and minor grooves.
a. Major groove – larger of two empty spaces in an
imaginary cylinder that encloses the DNA double
helix.
b. Minor groove – smaller of two empty spaces in an
imaginary cylinder that encloses the DNA double
helix
3. Each base is hydrogen bonded to a base in the opposite
strand to form a planar base pair. The Watson–Crick
structure can accommodate only two types of base pairs.
Each adenine residue must pair with a thymine residue and
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
64
Melting – or heat denaturation of DNA
observing the absorption of ultraviolet light.
As the DNA is heated and the strands separate, the
wavelength of absorption does not change, but the amount
of light absorbed increases (hyperchromicity)
Bases become unstacked as the DNA is denatured.
a way to obtain single-stranded DNA
The higher the percentage of G}C base pairs, the higher the
melting temperature of a DNA molecule.
Annealing – or renaturation of denatured DNA
separated strands can recombine and form the same base
pairs responsible for maintaining the double helix.
Chromatin – a complex of DNA and protein found in eukaryotic nuclei.
Histones – basic proteins found complexed to eukaryotic DNA.
five main types: H1, H2A, H2B, H3, and H4
contain large numbers of basic amino acid residues, such as
lysine and arginine.
can be modified by acetylation, methylation,
phosphorylation, and ubiquitinylation.
Ubiquitin – a protein involved in the degradation of other
proteins.
Nucleosome – a globular structure in chromatin in which DNA is
wrapped around an aggregate of histone molecules.
Spacer regions – “string” portions”; consist of DNA complexed to
some H1 histone and non-histone proteins.
RNA Is a Single-Stranded Nucleic Acid
RNA – primarily as single strands, which usually form compact
structures rather than loose extended chains.
RNA strand — which is identical to a DNA strand except for
the presence of 2′-OH groups and the substitution of uracil
for thymine—can base-pair with a complementary strand of
RNA or DNA.
A pairs with U (or T in DNA), and G with C.
Base pairing often occurs intramolecularly, giving rise to
stem–loop structures or, when loops interact with each
other, to more complex structures.
Roles of RNA
Transfer RNA – a single-stranded polynucleotide chain, between 73
and 94 nucleotide residues long, that generally has a molecular mass
of about 25,000 Da
where intrachain hydrogen bonding occurs, forming A–U
and G–C base
duplexes thus formed have the A-helical form
can be drawn as a cloverleaf structure
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
Biochemistry for Medical Laboratory Science
-
Stems – hydrogen-bonded portions of the molecule
Loops – non-hydrogen-bonded portions
bound to the ribosome in a definite spatial arrangement
that ensures the correct order of the amino acids in the
growing polypeptide chain.
After the tRNA is transcribed from DNA, a specific enzyme,
ATP(CTP): tRNA nucleotidyltransferase adds the sequence
CCA to the 3' end.
Ribosomal RNA – tend to be quite large, and only a few types exist in
a cell.
RNA portion of a ribosome accounts for 60% to 65% of the
total weight, and the protein portion constitutes the
remaining 35% to 40% of the weight.
Analytical ultracentrifugation – useful for monitoring the
dissociation and reassociation of ribosomes.
Sedimentation coefficient – motion of the particle;
expressed in Svedberg units (S), which are named after
Theodor Svedberg.
o S value increases with the molecular weight of the
sedimenting particle, but it is not directly
proportional to it because the particle’s shape
also affects its sedimentation rate.
Messenger RNA – least abundant of the main types of RNA
sequences of bases specify the order of the amino acids in
proteins.
formed when it is needed, directs the synthesis of proteins,
and then is degraded so that the nucleotides can be
recycled.
Heterogeneous nuclear RNA (hnRNA) – eukaryotic RNA
that is initially produced by transcribing DNA; it contains
intervening sequences that do not code for any proteins
Introns – intervening sequences in DNA that do not appear
in the final sequence of mRNA
protective units called 5'-caps and 3' poly(A) tails are added
before the mRNA is complete
Small Nuclear RNA – found in the nucleus of eukaryotic cells
about 100 to 200 nucleotides long.
it is complexed with proteins forming small nuclear
ribonucleoprotein particles, usually abbreviated snRNPs.
have a sedimentation coefficient of 10S.
help with the processing of the initial mRNA transcribed
from DNA into a mature form that is ready for export out of
the nucleus.
65
OVERVIEW OF NUCLEIC ACID FUNCTION
Double-stranded, or duplex, nature of DNA facilitates its replication.
When a cell divides, each DNA strand acts as a template for
the assembly of its complementary strand.
Each DNA molecule consists of one parental strand and one
daughter strand.
Daughter strands – synthesized by the stepwise
polymerization of nucleotides that specifically pair with
bases on the parental strands.
Genes Direct Protein Synthesis
RNA – link between DNA and enzymes
The DNA of a gene is transcribed to produce an RNA molecule that is
complementary to the DNA. The RNA sequence is then translated into
the corresponding sequence of amino acids to form a protein.
Central Dogma of Molecular biology – formulated by Crick in 1958

DNA directs its own replication to produce new DNA
molecules

DNA is transcribed into RNA

RNA is translated into proteins
1.
2.
3.
4.
5.
6.
7.
As the daughter strands of DNA are synthesized from free
deoxynucleoside triphosphates that pair with bases in the parent
DNA strand, RNA strands are synthesized from free
ribonucleoside triphosphates that pair with the complementary
bases in one DNA strand of a gene.
The RNA that corresponds to a protein-coding gene (called
messenger RNA, or mRNA) makes its way to a ribosome, an
organelle that is itself composed largely of RNA (ribosomal RNA,
or rRNA).
At the ribosome, each set of three nucleotides in the mRNA pairs
with three complementary nucleotides in a small RNA molecule
called transfer RNA, or tRNA. Attached to each tRNA molecule is
its corresponding amino acid.
The ribosome catalyzes the joining of amino acids.
Amino acids are added to the growing protein chain according to
the order in which the tRNA molecules bind to the mRNA.
Since the nucleotide sequence of the mRNA in turn reflects the
sequences of nucleotides in the gene, DNA directs the synthesis
of proteins.
DNA follows that alterations to the genetic material of an
organism (mutations) may manifest themselves as proteins with
altered structures and functions.
Genomics – study of the genome’s size, organization, and gene content
Transcriptomics – study of gene expression, which focuses on the set
of mRNA molecules, or transcriptome, that is transcribed from DNA
under any particular set of circumstances.
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Biochemistry for Medical Laboratory Science
Proteomics – study of the proteins (the proteome) produced as a result
of transcription and translation.
BIOSYNTHESIS OF NUCLEIC ACIDS: REPLICATION
Replication – the process of duplication of DNA
Transcription – the process of formation of RNA on a DNA template
Translation – the process of protein synthesis in which the amino acid
sequence of the protein reflects the sequence of bases in the gene
that codes for that protein
Reverse transcriptase – the enzyme that directs the synthesis of DNA
on an RNA template
Important challenges:
1. Separating the two DNA strands
two strands of DNA are wound around each other in such a
way that they must be unwound if they are to be separated.
achieving continuous unwinding of the double helix, the cell
also must protect the unwound portions of DNA from the
action of nucleases that attack single-stranded DNA.
2. Synthesizing of DNA from the 5' to the 3' end
the template has one 5' S 3' strand and one 3' S 5' strand, as
does the newly synthesized DNA.
3. Guarding against errors in replication
ensuring that the correct base is added to the growing
polynucleotide chain.
66
Density-gradient centrifugation – the technique of separating
substances in an ultracentrifuge by applying the sample to the top of
a tube that contains a solution of varying densities.
2. Bidirectional – occurs in two directions about the origin of
replication.
Origin of replication – the point at which the DNA double
helix begins to unwind at the start of replication.
Replication forks – in DNA replication, the points at which
new DNA strands are formed.
 structure – a bubble (also called an “eye”) of newly
synthesized DNA between regions of the original DNA is a
manifestation of the advance of the two replication forks in
opposite directions.
new polynucleotide chains are synthesized in the 5' S 3'
direction.
3. Semidiscontinuous DNA Replication – the lagging chain is first
made as short chains (Okazaki fragments) which are subsequently
linked to form long chains while the leading strand is formed in a
continuous manner.
Characteristics of DNA Replication
1. Semiconservative Replication – the mode in which DNA
reproduces itself, such that one strand comes from parent DNA
and the other strand is newly formed.
Proteins required for DNA Replication
DNA gyrase – an enzyme that introduces supercoiling into closed
circular DNA.
catalyzes the conversion of relaxed, circular DNA with a nick
in one strand to the supercoiled form with the nick sealed
that is found in normal prokaryotic DNA which introduces
supercoiling.
fights positive supercoils by putting negative super- coils
ahead of the replication fork.
Helicase – promotes unwinding by binding at the replication fork.
Single-strand binding protein (SSB) – protects exposed single- strand
sections of DNA from hydrolysis by nucleases.
Primase – makes a short section of RNA to act as a primer for DNA
synthesis
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Biochemistry for Medical Laboratory Science
-
responsible for copying a short stretch of the DNA template
strand to pro- duce the RNA primer sequence.
Primosome – the complex at the replication fork in DNA
synthesis; it consists of the RNA primer, primase, and
helicase
DNA polymerase III – starts the synthesis of two new strands of DNA
newly formed DNA is linked to the 3'-hydroxyl of the RNA
primer, and synthesis proceeds from the 5' end to the 3' end
on both the leading and the lagging strands.
As the replication fork moves, the RNA primer is removed by
polymerase I, using its exonuclease activity.
The primer is replaced by deoxynucleotides, also by DNA polymerase
I, using its polymerase activity.
-
67
Proofreading and Repair
Proofreading – removal of incorrect nucleotides immediately after
they are added to the growing DNA during the replication process.
Two major fragments of Pol I:
a. Klenow fragment – contains the polymerase activity and
the proofreading activity.
b. other contains the 5'  3' repair activity
a dimer of the  -subunit, and it forms a closed ring, called
a sliding clamp, around the DNA chain
Nick Translation – a type of DNA repair that involves polymerase I
using its 5' to 3' exonuclease activity to remove primers or replace
damaged nucleotides.
Mutagens – agents that bring about a mutation; such
agents include radiation and chemical substances that alter
DNA
Mismatch repair – a type of DNA repair that begins when repair
enzymes find two bases that are incorrectly paired.
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Paulo Coelho
Biochemistry for Medical Laboratory Science
-
area with the mismatch is removed, and DNA polymerases
replicate the area again.
-
Base-Excision Repair – a type of DNA repair that begins with an
enzyme removing a damaged base, followed by removal of the rest of
the nucleotide.
A base that has been damaged by oxidation or chemical
modification is removed by DNA glycosylase, leaving an AP
site.
AP endonuclease – removes the sugar and phosphate from
the nucleotide.
Excision exonuclease – removes several more bases.
DNA polymerase I – fills in the gap.
DNA ligase – seals the phosphodiester backbone.
68
common for DNA lesions caused by ultraviolet or chemical
means, which often lead to deformed DNA structures.
DNA polymerase I and DNA ligase – work to fill in the gap
Eukaryotic DNA Replication
More complicated in three basic ways:
a. there are multiple origins of replication
b. the timing must be controlled to that of cell divisions
c. more proteins and enzymes are involved.
DNA replication takes place during a few hours in the S phase, and
pathways exist to make sure that the DNA is replicated only once per
cycle.
Replicators – the multiple origins of replication in eukaryotic DNA
synthesis.
Replicons – parts of chromosomes in which DNA synthesis is taking
place.
Replication and Cell Division
Origin recognition complex (ORC) – a protein complex bound to DNA
throughout the cell cycle that serves as an attachment site for several
proteins that help control replication.
Replication activator protein (RAP) – the protein whose binding
prepares for the start of DNA replication in eukaryotes.
Replication licensing factors (RLFs) – proteins required for DNA
replication in eukaryotes.
Nucleotide-Excision Repair – a type of DNA repair in which damaged
or deformed DNA is repaired by removal of a section of DNA
containing the damage.
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Biochemistry for Medical Laboratory Science
69
Pre-replication complex (pre-RC) – the complex of DNA, recognition
protein (ORC), activator protein (RAP), and licensing factors (RLFs)
that makes DNA competent for replication in eukaryotes.
Cyclins – proteins that play an important role in control of the cell
cycle by regulating the activity of kinases.
Cyclin-dependent protein kinases – protein kinases that interact with
cyclins and control replication.
DNA replication in eukaryotes is semiconservative.
There is a leading strand with continuous synthesis in the 5'
S 3' direction and a lagging strand with discontinuous
synthesis in the 5' S 3' direction.
Pol  - associated with primase activity
Initiated formation of Okazaki fragments (150 to 200
nucleotides long)
Dissociates and is replaced by Pol and its attached PCNA
protein.
RFC (replication factor C) – involved in attaching PCNA to Pol .
FEN-1 and RNase H1 – degrade the RNA.
Pol  – fills in the gaps made by primer removal.
Eukaryotic DNA Polymerases
, , , and  enzymes – found in the nucleus,
 form – occurs in mitochondria.
Polymerase  – first discovered, and it has the most subunits.
has the ability to make primers, but it lacks a 3' S 5'
proofreading activity and has low processivity.
Polymerase  – the principal DNA polymerase in eukaryotes.
interacts with a special protein called PCNA (for
proliferating cell nuclear antigen)
PCNA – the eukaryotic equivalent of the part of Pol III that
functions as a sliding clamp ()
Polymerase  – involved in leading strand replication.
TRANSCRIPTION OF THE GENETIC CODE: THE BIOSYNTHESIS OF RNA
Transcription – the process of formation of RNA on a DNA template.
major control point in the expression of genes and the
production of proteins.
may replace polymerase  in lagging strand synthesis.
Polymerase  – appears to be a repair enzyme.
Polymerase  – carries out DNA replication in mitochondria.
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Paulo Coelho
Biochemistry for Medical Laboratory Science
-
produces all types of RNA: mRNA, tRNA, rRNA, snRNA,
miRNA, and siRNA.
TRANSCRIPTION IN PROKARYOTES
RNA polymerase – the enzyme that catalyzes the production of RNA
on a DNA template.
Five different types of subunits: , , , ’, and 
Actual composition: 2’
-subunit – involved in the recognition of specific
promoters.
-, ’-, -, and -subunits – combine to make the active site
for polymerization.
Core enzyme – the enzyme lacking the sigma subunit (2’).
Holoenzyme – an enzyme that has all component parts, including
coenzymes and all subunits.
Template strand (antisense, or [ - ] strand) – the DNA strand that is
used as a template for RNA synthesis (strand)
strand that directs the synthesis of the RNA
its code is the complement of the RNA that is produced.
Coding strand (sense, [+], or non-template strand) – the DNA strand
that has the same sequence as the RNA that is synthesized from the
template (strand).
RNA sequence used to determine what amino acids are
produced in the case of mRNA.
Promoter – the portion of DNA to which RNA polymerase binds at the
start of transcription.
Transcription start site (TSS) – the location on the template DNA strand where the first ribonucleotide is used to initiate RNA synthesis.
first base to be incorporated into the RNA chain at position
+1.
Pribnow box – a DNA base sequence that is part of a prokaryotic
promoter; it is located 10 bases before the transcription start site.
-10 region, about 10 bases upstream.
70
-35 region (-35 element) – a portion of DNA that is 35 base pairs
upstream from the start of RNA transcription that is important in
control of RNA synthesis in bacteria.
Core promoter – in prokaryotic transcription, the portion of the DNA
from the transcription start site to the -35 region.
UP element – a prokaryotic promoter element that is 40 to 60 bases
upstream of the transcription start site.
enhances the binding of RNA polymerase.
Extended promoter – in prokaryotic transcription, the DNA from the
transcription start site to the UP element.
Consensus sequences – DNA sequences to which RNA polymerase
binds; they are identical in many organisms.
Chain initiation – the part of transcription where RNA polymerase
binds to DNA, the strands are separated, and the first nucleotide binds
to its complement.
first phase of transcription.
most controlled.
1. Closed complex – the complex that initially forms between RNA
polymerase and DNA before transcription begins.
o -subunit directs the polymerase to the promoter. It
bridges the 210 and 235 regions of the promoter to the RNA
polymerase core via a flexible “flap” in the --subunit.
o Core enzymes lacking the --subunit bind to areas of DNA
that lack promoters. The holoenzyme may bind to
“promoterless” DNA, but it dissociates without transcribing.
2. Open complex – the form of the complex of RNA polymerase and
DNA that occurs during transcription.
o a portion of the ' and the -subunits initiate strand
separation, melting about 14 base pairs surrounding the
transcription start site.
o A purine ribonucleoside triphosphate is the first base in
RNA, and it binds to its complementary DNA base at
position 11.
o Of the purines, A tends to occur more often than G. This first
residue retains its 5'-triphosphate group
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Biochemistry for Medical Laboratory Science
71
- It “scrunches” the DNA into itself, causing torsional strain of
the separated DNA strands. Like a bow loading up with
potential energy as the bowstring is pulled, this provides
the energy to allow the polymerase to break free.
Inverted repeats – terminate transcription. Inverted repeats in the
DNA sequence being transcribed can lead to an mRNA molecule that
forms a hairpin loop. This is often used to terminate transcription.
Two types of Termination Mechanisms:
1. Intrinsic termination – the type of transcription termination that is
not dependent on the rho protein
Termination sites – the areas in DNA that cause termination
of transcription by generating hairpin loops and a zone of
weak binding between DNA and RNA.
Inverted repeats are sequences of bases that are
complementary.
2. Rho () -dependent termination – also cause a hairpin loop to form
 protein binds to the RNA and chases the polymerase.
 protein reaches the termination site, it facilitates the
dissociation of the transcription machinery.
After the strands have separated, a transcription bubble of about 17
base pairs moves down the DNA sequence to be transcribed.
RNA polymerase catalyzes the formation of the phosphodiester
bonds between the incorporated ribonucleotides.
When about 10 nucleotides have been incorporated, the -subunit
dissociates and is later recycled to bind to another RNA polymerase
core enzyme.
Transcription process supercoils DNA, with negative supercoiling upstream of the transcription bubble and positive supercoiling
downstream.
Abortive transcription – the failure of RNA polymerase to break its
own bonds to the promoter via the -subunit.
The RNA polymerase is bound tightly to the DNA promoter.
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Biochemistry for Medical Laboratory Science
72
Transcription Regulation in Prokaryotes
Control of transcription via different  subunits:
A. When the phage SPO1 infects B. subtilis, the host RNA
polymerase (tan) and s-subunit (blue) transcribe the early genes
of the infecting viral DNA. One of the early gene products is
gp28 (green), an alternative -subunit.
B. The gp28 directs the RNA polymerase to transcribe the middle
genes, which produces gp33 (purple) and gp34 (red).
C. The gp33 and gp34 direct the host’s RNA polymerase to
transcribe the late genes.
Enhancers – DNA sequences that bind to a transcription factor and
increase the rate of transcription.
- polymerase does not bind to enhancers.
Transcription factors – proteins or other complexes that bind to DNA
sequences and alter the basal level of transcription.
Response elements – DNA sequences that bind to transcription
factors involved in more generalized control of pathways.
Silencer – a DNA sequence that binds to a transcription factor and
reduces the level of transcription.
Operon – a group of operator, promoter, and structural genes inducer
a molecule that turns on the transcription of a gene.
these proteins can be triggered by the presence of a
suitable substance called an inducer
Induction – (of enzyme synthesis) the triggering of the production of
an enzyme by the presence of a specific inducer.
Structural gene – a gene that directs the synthesis of a protein under
the control of some regulatory gene.
Regulatory gene – a gene that directs the synthesis of a repressor
protein.
Repressor – a protein that binds to an operator gene, blocking the
transcription and eventual translation of structural genes under the
control of that operator.
Operator – the DNA element to which a repressor of protein synthesis
binds.
Control sites – the operator and promoter elements that modulate
the production of proteins whose amino acid sequence is specified by
the structural genes under their control.
Catabolite repression – repression of the synthesis of lac proteins by
glucose.
Catabolite activator protein (CAP) – a protein that can bind to a
promoter when complexed with cAMP, allowing RNA polymerase to
bind to its entry site on the same promoter.
The cell has an adequate supply of glucose, the level of cAMP is low.
It takes the presence of lactose and the absence of glucose for the
operon to be active.
Inducible (and/or repressible) – describes an operon whose gene
expression is controlled by the presence or absence of an inducer or
a repressor.
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Paulo Coelho
Biochemistry for Medical Laboratory Science
Co-inducer – a small molecule inside an inducible operon that binds
to an inducer or a repressor.
Constitutive – refers to the transcription and expression of genes that
are not controlled by anything other than the inherent binding of the
RNA polymerase to the promoter.
Co-repressor – a substance that binds to a repressor protein, making
it active and able to bind to an operator gene
73
4. Downstream element
Initiation of Transcription
Preinitiation complex – in eukaryotic transcription, the phase where
RNA Polymerase and the general transcription factors bind to the
DNA.
normally contains RNA polymerase II and six general
transcription factors (GTFs)
General transcription factors (GTFs) – the six transcription factors
that first bind to DNA to initiate transcription.
TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.
TRANSCRIPTION IN EUKARYOTES
Three primary RNA polymerases:
1. RNA polymerase I – found in the nucleolus and synthesizes
precursors of most, but not all, ribosomal RNAs.
2. RNA polymerase II – found in the nucleoplasm and synthesizes
mRNA precursors.
3. RNA polymerase III – found in the nucleoplasm and synthesizes the
tRNAs, precursors of 5S ribosomal RNA, and a variety of other small
RNA molecules involved in mRNA processing and protein transport.
Structure of RNA Polymerase II
RNA polymerase II – the most extensively studied,
yeast Saccharomyces cerevisiaie is the most common model
system.
C-terminal domain (CTD) – the region of a protein at the C-terminus,
especially important in eukaryotic RNA polymerase B.
Pol II Promoters
1. Upstream elements – in transcription, a portion of the sequences
closer to the 3' end than the gene to be transcribed, where the DNA
is read from the 3' to the 5' end and the RNA is formed from the 5' to
the 3' end; in translation, nearer to the 5' end of the mRNA.
act as enhancers and silencers.
Two common elements that are close to the core promoter:
GC box (-40), which has a consensus sequence of GGGCGG,
and the CAAT box (extending to -110), which has a
consensus sequence of GGCCAATCT.
2. TATA box – a promoter element found in eukaryotic transcription
that is located 25 bases upstream of the transcription start site.
has a consensus sequence of TATAA(T/A).
3. Initiator element (Inr) – a loosely conserved sequence surrounding
the transcription start site in eukaryotic DNA.
Sequence of events in Pol II transcription:
1. Recognition of the TATA box by TFIID. This transcription factor is
actually a combination of several proteins. The primary protein is
called TATA-binding protein (TBP) with many TBP-associated factors
(TAFIIs)
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Biochemistry for Medical Laboratory Science
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TBP – universal transcription factor.
2. The TBP protein binds to the minor groove of the DNA at the TATA
box via the last 180 amino acids of its C-terminal domain. The minor
groove of the DNA is opened, and the DNA is bent to an 80° angle.
3. Once TFIID is bound, TFIIA binds, and TFIIA also interacts with both
the DNA and TFIID. TFIIB also binds to TFIID, bridging the TBP and Pol
II.
4. TFIIF then binds tightly to Pol II and suppresses nonspecific binding.
Pol II and TFIIF then bind stably to the promoter. TFIIF interacts with
Pol II, TBP, TFIIB, and the TAFIIs. It also regulates the activity of the
CTD phosphatase.
5. TFIIE interacts with un- phosphorylated Pol II.
6. In the open complex, the Pol II CTD is phosphorylated, and the DNA
strands are separated
Elongation and Termination of Transcription
Phosphorylated Pol II synthesizes RNA and leaves the promoter
region behind. At the same time, the GTFs either are left at the
promoter or dissociate from Pol II.
TFFIF – has a separate stimulatory effect on elongation.
promotes a rapid read-through of pause sites, perhaps
locking the Pol II into an elongation-competent form that
does not pause and dissociate.
TFIIS – second elongation factor.
are called arrest release factors.
help the RNA polymerase move again after it has paused.
P-TEF and N-TEF proteins (positive-transcription elongation factor
and negative-transcription elongation factor) – third class of
elongation factors
increase the productive form of transcription and decrease
the abortive form, or vice versa.
Elongation is controlled in several ways:
a. There are sequences called pause sites, where the RNA
polymerase hesitates.
b. Elongation can be aborted, leading to premature
termination.
c. Elongation can proceed past the normal termination point.
This is called antitermination.
Eukaryotic consensus sequence for termination: AAUAAA.
may be 100 to 1000 bases away from the actual end of the
mRNA.
Transcription Regulation in Eukaryotes
Basal level – (of transcription) the level of transcription that occurs
due solely to RNA polymerase and the general transcription factors
Activators – (of transcription) molecules that raise the level of
transcription above the basal level
Mediator – a giant protein complex that bridges the promoter,
general transcription factors and remote silencers and enhancers.
a crescent-shaped protein with a head, middle, and tail.
Chromatin remodeling complexes – enzyme complexes that mediate
ATP-dependent conformational changes in nucleosome structure
that lead to transcription.
huge (1 megadalton) assemblies containing ATP-dependent
enzymes that loosen the DNA: protein interactions in
nucleosomes by a variety of mechanisms involving sliding,
ejecting, inserting, and otherwise restructuring the core
octamers.
create space between the nucleosomes and to expose the
DNA so that RNA polymerase can be recruited to the
promoter.
Histone-modifying enzymes – enzymes that make covalent
modifications to the histone core octamer.
Histone acetyltransferases (HATs) – enzymes that acetylate lysine
residues on histone proteins.
acetylation of the -amino groups of lysine on the histone
tails
removes the positive charge and loosens the binding of the
DNA.
Histone deacetylase (HDAC) – an enzyme that removes the acetyl
group from an acetylated lysine on a histone protein.
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Biochemistry for Medical Laboratory Science
Histone code – a term for the combination of events revolving around
chromatin remodeling that control transcription.
Response elements: heat-shock element (HSE), the glucocorticoidresponse element (GRE), the metal-response element (MRE), and the
cyclic-AMP-response element (CRE).
Cyclic-AMP-response element (CRE) – an important eukaryotic
response element that is controlled by production of cAMP in the cell.
Cyclic AMP – produced as a second messenger from several
hormones, such as epinephrine and glucagon.
levels of cAMP rise, the activity of cAMP-dependent
protein kinase (protein kinase A) is stimulated.
phosphorylates many other proteins and enzymes inside
the cell and is usually associated with switching the cell to a
catabolic mode, in which macromolecules are broken down
for energy.
Protein kinase A – phosphorylates a protein called cyclic-AMPresponse-element binding protein (CREB), which binds to the cyclicAMP-response element and activates the associated genes.
CREB-binding protein (CBP) – an important mediator of transcription,
it links the basal transcription machinery to CREB.
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75
Biochemistry for Medical Laboratory Science
PROTEIN SYNTHESIS: TRANSLATION OF THE GENETIC MESSAGE
Protein biosynthesis – a complex process requiring ribosomes,
messenger RNA (mRNA), transfer RNA (tRNA), and a number of
protein factors.
o Ribosome – the site of protein synthesis.
o mRNA and tRNA – bound to the ribosome in the course of
protein synthesis, are responsible for the correct order of
amino acids in the growing protein chain.
Activated – (in protein synthesis) describes a process in protein
synthesis by which an amino acid is bonded to tRNA.
Aminoacyl-tRNA synthetases – enzymes that catalyze the formation
of an ester linkage between an amino acid and tRNA.
Three steps of formation of the polypeptide chain:
1. Chain initiation – the binding of the first aminoacyl-tRNA to the
start site on the ribosome.
first aminoacyl-tRNA is bound to the mRNA at the site that
encodes the start of polypeptide synthesis.
next aminoacyl-tRNA forms a complex with the ribosome
and with mRNA.
2. Chain elongation – the formation of peptide bonds between
successive amino acid residues.
repeats itself until the poly- peptide chain is complete.
3. Chain termination – the release of a newly formed protein from
the ribosome
76
Start signal – a mRNA triplet that begins the sequence that directs
polypeptide synthesis.
Initiation complex – the aggregate of mRNA, N-formylmethionetRNA, ribosomal subunits, and initiation factors needed at the start of
protein synthesis.
eight components enter into the formation of the fmet
initiation complex, including mRNA, the 30S ribosomal
subunit, fmet-tRNAfmet, GTP, and three protein initiation
factors, called IF-1, IF-2, and IF-3. The IF-3 protein facilitates
the binding of mRNA to the 30S ribosomal subunit
appears to prevent premature binding of the 50S subunit,
which takes place in a subsequent step of the initiation
process.
The formation of an initiation complex:
The 30S ribosomal subunit binds to mRNA and fmet-tRNAfmet in the
presence of GTP and the three initiation factors, IF-1, IF-2, and IF-3,
forming the 30S initiation complex. The 50S ribosomal subunit is
added, forming the 70S initiation complex.
The Genetic Code
Genetic message – contained in a triplet, nonoverlapping,
commaless, degenerate, universal code.
a. Triplet code (codon) – a sequence of three bases (a triplet)
in mRNA that specifies one amino acid in a protein.
b. Nonoverlapping – indicates that no bases are shared
between consecutive codons; the ribosome moves along
the mRNA three bases at a time rather than one or two at a
time.
c. Commaless – no intervening bases exist between codons.
d. Degenerate code – more than one triplet can encode the
same amino acid; acts as a buffer against deleterious
mutations.
PROKARYOTIC TRANSLATION
Chain Initiation
N-formylmethionine (fmet) – initial N-terminal amino acid of all
proteins.
Two tRNAs for methionine in E. coli: one for unmodified methionine
(tRNAmet) and one for N-formylmethionine (tRNAfmet).
met-tRNAmet and met-tRNAfmet – aminoacyl-tRNAs that they form
with methionine.
N-formylmethionine-tRNAfmet (fmet-tRNAfmet) – a formylation
reaction takes place after methionine is bonded to the tRNA.
30S initiation complex – the combination of mRNA, aminoacyl-tRNA,
and 30S ribosomal subunit.
70S initiation complex – a 30S initiation complex plus a 50S ribosomal
subunit
Shine–Dalgarno sequence – a purine-rich leader sequence in
prokaryotic mRNA that precedes the start signal (5'-GGAGGU-3')
usually lies about 10 nucleotides upstream of the AUG start
signal
acts as a ribosomal binding site
also known as the initiation codon
binds to a pyrimidine-rich sequence on the 16S ribosomal
RNA part of the 30S subunit and aligns it for proper
translation beginning with the AUG start codon.
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Biochemistry for Medical Laboratory Science
Chain Elongation
Three tRNA binding sites:
a. P (peptidyl) site – binds a tRNA that carries a peptide chain
b. A (aminoacyl) site – binds an incoming aminoacyl-tRNA
c. E (exit) site – carries an uncharged tRNA that is about to be
released from the ribosome
Step 1: An aminoacyl-tRNA is bound to the A site on the ribosome.
Elongation factor EF-Tu (Tu) and GTP are required. The P site on the
ribosome is already occupied.
Step 2: Elongation factor EF-Tu is released from the ribosome and
regenerated in a process requiring elongation factor EF-Ts (Ts) and
GTP.
Step 3: The peptide bond is formed, leaving an uncharged tRNA at the
P site.
Step 4: In the translocation step, the uncharged tRNA is released. The
peptidyl-tRNA is translocated to the P site, leaving an empty A site.
The uncharged tRNA is translocated to the E site and subsequently
released. Elongation factor EF-G and GTP are required.
Chain Termination
UAA, UAG, and UGA – the stop signals
not recognized by any tRNAs, but they are recognized by
proteins called release factors.
1. GTP, which is bound to a third release factor, RF-3.
2. RF-1 binds to UAA and UAG, and RF-2 binds to UAA and UGA.
3. RF-3 does not bind to any codon, but it does facilitate the activity
of the other two release factors.
4. Either RF-1 or RF-2 is bound near the A site of the ribosome when
one of the termination codons is reached.
5. Conserved sequence of Gly-Gly-Gln is essential for the hydrolysis
reaction of the RF. GTP is hydrolyzed in the process.
6. Whole complex dissociates, setting free the release factors, tRNA,
mRNA, and the 30S and 50S ribosomal subunits.
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho
77
Biochemistry for Medical Laboratory Science
78
that of miRNA-430 in zebrafish, the primary effect of the miRNA is to
EUKARYOTIC TRANSLATION
Chain Initiation
Eukaryotic initiation factor (eIF) – a protein involved in the initiation
inhibit the initiation of translation of the corresponding mRNA.
Chain Elongation
-
of translation in eukaryotes.
Step 1. Chain initiation involves the assembly of a 43S preinitiation
complex. The initial amino acid is usually methionine, which is
attached to a special tRNAi that serves only as the initiator tRNA.
There is no fmet in eukaryotes. The met-tRNAi is delivered to the 40S
ribosomal subunit as a complex with GTP and eIF2. The 40S ribosome
is also bound to eIF1A and eIF3. This order of events is different from
that in prokaryotes in that the first tRNA binds to the ribosome
without the presence of the mRNA.
Step 2. The mRNA is recruited. There is no Shine–Dalgarno sequence
for location of the start codon. The 5' cap orients the ribosome to the
correct AUG via what is called a scanning mechanism, which is driven
by ATP hydrolysis. The eIF4E is also a cap-binding protein, which forms
a complex with several other eIFs. A poly A binding protein (Pab1p)
links the poly A tail to eIF4G. The eIF-40S com- plex is initially
positioned upstream of the start codon (Figure 12.23). It moves
downstream until it encounters the first AUG in the correct context.
The con- text is determined by a few bases surrounding the start
codon, called the Kozak sequence. It is characterized by the consensus
sequence 23ACCAUGG14. The ribosome may skip the first AUG it finds
if the next one has the Kozak sequence, although the AUG closest to
the 5' end of the mRNA is usually the start codon. Another factor is
the presence of mRNA secondary structure. If hairpin loops form
downstream of an AUG, an earlier AUG may be chosen. The mRNA
and the seven eIFs constitute the 48S preinitiation complex.
very similar to that of prokaryotes.
same mechanism of peptidyl transferase and ribosome
translocation
Two eukaryotic elongation factors:
1. eEF1 consists of two subunits, eEF1A and eEF1B.
a. 1A subunit is the counterpart of EF-Tu in prokaryotes.
b. 1B subunit is the equivalent of the EF-Ts in prokaryotes.
2. eEF2 protein is the counterpart of the prokaryotic EF-G, which
causes translocation.
Antibiotic chloramphenicol – (a trade name is Chloromycetin) binds
to the A site and inhibits peptidyl transferase activity in prokaryotes,
but not in eukaryotes.
- useful in treating bacterial infections.
Diphtheria toxin – (in eukaryotes) a protein that interferes with
protein synthesis by decreasing the activity of the eukaryotic
elongation factor eEF2.
Chain Termination
Ribosome encounters a stop codon, either UAG, UAA, or UGA.
- only one release factor binds to all three stop codons and
catalyzes the hydrolysis of the bond between the C-terminal
amino acid and the tRNA.
Suppressor tRNA – allows translation to continue through a stop
codon. Suppressor tRNAs tend to be found in cells in which a mutation
has introduced a stop codon.
Step 3. The 60S ribosome is recruited, forming the 80S initiation
complex. GTP is hydrolyzed, and the initiation factors are released.
The initiation of eukaryotic translation is also a control point in overall
gene expression. In the last chapter we looked at the effects of miRNA
on gene expression, focusing mainly on how transcription of mRNA
was affected. Recent studies have indicated that in at least one case,
“Whatever you decide to do, make sure it makes you happy.”
Paulo Coelho