UNIVERSITI TEKNOLOGI MARA
FACULTY OF HEALTH SCIENCES
BIOCHEMISTRY FOR ENVIRONMENTAL HEALTH
INSTITUT PERKEMBANGAN PENDIDIKAN (InED)
UNIVERSITI TEKNOLOGI MARA (UiTM)
40450 SHAH ALAM
STUDY GUIDE for BIOCHEMISTRY
2009
BIOCHEMISTRY FOR ENVIRONMENTAL HEALTH
(ENV 400/416)
Bachelor in Environmental Safety and Health
(Honours) Program (e- pjj)
Faculty of Health Sciences
Universiti Teknologi Mara (UiTM)
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STUDY GUIDE for BIOCHEMISTRY
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Course Description:
This is an introduction to the chemistry of biological compounds. A systematic study of
carbohydrates, lipids, amino-acids, proteins, nucleic acids, and their components is
presented. Metabolism of the biological compounds is also studied as are the
interrelations among the carbon, nitrogen, and energy cycles. Enzymology, intermediary
metabolism, and metabolic control will also be included.
Course Outcomes:
Upon successful completion of this course the student should be able to:
1.
Explain the different types of bonding and interactions found in biochemistry such
as hydrogen bonds, ionic bonds, hydrophobic interaction, Van der Waals forces
and asymmetry of carbon compounds with cis-trans isomerism.
2.
Explain that water molecules are polar and form irregular hydrogen-bonded
networks in liquid state and why polar and ionic substances dissolve in water.
3.
Explain how acids and bases affect the pH of a solution, the relationship between
pH and pK for a solution of weak acid and how the buffer works.
4.
Describe the structure of an amino acid and the structure of the 20 different R
groups.
5.
Describe the structure of alpha helix, beta sheet primary, secondary, tertiary and
quartenary and the covalent and non-covalent forces that maintain structures.
6.
Describe the metabolic disorder, phenyketonuria.
7.
Explain how the Michaelis-Menten equation relates the initial velocity of a
reaction for an enzyme substrate reaction and Lineweaver plot can be used to
present kinetic data.
8.
Describe competitive and non-competitive inhibitor.
9.
Explain the common features in amino acid biosynthesis and the role of
urea cycle in amino acid breakdown.
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10.
Explain the levels of nucleic acid structure and the structure and functions of DNA
and RNA
11..
Describe how monosaccharide cyclize to from two different anomers and the
monosaccharide linkages in polysaccharides.
12.
Describe lactose intolerance, diabetes and hypoglycemia
13.
Describe glycolysis and the citric acid cycle to synthesize ATP and some allied
health perspective of anerobic metabolism with dental plaque.
14.
Describe electron carriers and how electrons travel from the different complexes.
15.
Describe the connection between Electron Transport Chain and Oxidative
Phosphorylation.
16.
Describe the structure and nomenclature of lipids including fatty acids,
triacyglycerols, sphingolipids and phophoglycerides.
17.
Explain the physiological roles of lipids as membrane components and energy
storage molecules.
18.
Explain lipid of lung surfactant.
19.
Explain fatty acid synthesis and degradation.
20.
Explain cholesterol biosynthesis and atherosclerosis
.
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STUDY GUIDE for BIOCHEMISTRY
CONTENTS
1.0
2009
PAGE
Basic Aspects of the chemistry of life
1.1
Biochemistry as the chemistry of living systems
1.2
Asymmetry of carbon compounds and cis-trans isomerism
1.3
Different types of bonding such as hydrogen bonds, ionic bonds
hydrophobic interactions, Van der Waals forces
2.0
Water, acid and base, buffer
3.0
2.1
Physical properties of water
2.2
Biological importance of water as a solvent
2.3
Hydrogen ion concentration and pH of biological systems
2.4
Relationship between pH and pK for a solution of weak acid
2.5
Physiological buffer systems
Amino acids and Proteins
3.1
Overall structure and properties of the 20 different R groups.
3.2
Ionizable groups in amino acids.
3.3
Peptide bonds link amino acid residues in a polypeptide
3.4
The structure of primary, secondary, tertiary and quartenary proteins and
the covalent and non-covalent forces that maintain structures.
3.5
4.0
The metabolic disorder, phenyketonuria
Properties of Enzymes
4.1
Classification and general catalytic properties of enzymes.
4.2
Michaelis-Menten equation relates to the initial velocity of a reaction for an
enzyme substrate reaction
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4.3
Lineweaver plot to present kinetic data.
4.4
Competitive and non-competitive inhibitor and examples.
STUDY GUIDE for BIOCHEMISTRY
5.0
2009
Nitrogen metabolism
6.0
5.1
Common features in amino acid metabolism
5.2
Glucogenic and Ketogenic amino acids
5.3
The role of urea cycle in amino acid breakdown
Sugar and carbohydrate structure
6.1
Monosaccharide and their derivates.
6.2
Cyclization to from two different anomers and glycosidic bond that links
two monossacrides.
7.0
6.3
Dissacharides and other sugars example as sweeteners
6.4
Polisaccharides such as starches and glycogen, cellulose.
6.5
Lactose intolerance, diabetes , hyphoglyceamia and hyperglycemia.
Metabolic processes central to ATP synthesis- Glycolysis and Citric acid
cycle
7.1
Glycolysis involves the breakdown of glucose to pyruvate to synthesize
ATP
7.2
Aerobic and anaerobic metabolism.
7.3
The citric acid cycle, a multistep catalytic process that converts acetyl
groups to NADH, FADH and GTP.
7.4
8.0
Allied health perspective of anaerobic metabolism
Electron transport and oxidative phosphorylation
8.1
Electron carriers as electrons travel from the different complexes.
8.2
The reactions catalyzed by the complexes and their mechanism
8.3
The connection between electron transport chain
phosphorylation.
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and
oxidative
STUDY GUIDE for BIOCHEMISTRY
9.0
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Lipid and Membranes
9.1
Structure and nomenclature of lipids including fatty acids, triacyglycerols,
sphingolipids and phophoglycerides
9.2
the physiological roles of lipids as membrane components and energy
storage molecules.
9.3
10.0
Lipid of lung surfactant
Lipid metabolism
11.0
10.1
Steps of fatty acid synthesis and its mode.
10.2
HMG-CoA is important in cholesterol biosynthesis.
10.3
Atherosclerosis
Nucleotides, nucleic acids
11.1
Levels of nucleic acid structure – nitrogenous bases, nucleosides,
nucleotides.
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11.2
Structure and functions of DNA and RNA .
11.3
Use of nucleoside analogues as drugs
STUDY GUIDE for BIOCHEMISTRY
2009
SYLLABUS CONTENTS
CHAPTER 1 : Basic aspects of the chemistry of life
1.1
biochemistry is the chemistry of living systems:
1. complicated and highly organized
2. each part has a function
3. function is related to structure
4. must extract energy from the environment
chemicals of living systems
•
alcohols
•
esters
•
ethers
•
amides
•
acids
•
anhydrides
•
also include thiols and phosphates
Biochemistry deals with the structure and function of biomolecules
biochemists study the structures of bio-molecules and their cellular
functions to better understand living systems and their chemistry
Example of structure-function relationship
1. amino acids are joined to form proteins and these proteins fold up to form
functional enzymes
2. nucleotides are joined to form Rna and Dna. these polymers are the
information molecules of living systems and maintain the genetic heritage
of organisms
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3. proteins (enzymes), RNA and DNA along with other molecules aggregate
to form cellular components, cells, organs and whole organisms.
•
Rna comes in 3 basic forms:
•
tRNA (transfer rna) = adapter in protein synthesis - matches codon to
amino acid
•
Rrna (ribosomal RNA) = structural RNA in ribosomes
•
mRNA (messenger rna) = contains information for protein synthesis
cell structure
basics of the relationship between proteins and DNA:
•
linear relationship between DNA, RNA and protein sequence
•
DNA encodes amino acids of a protein using 3 letter codons.
•
DNA is transcribed to make mRNA.
mRNA is translated by ribosomes to make the protein.
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Biochemistry can be divided into 3 areas of study
•
conformational- structure and 3d arrangements of biomolecules
•
metabolism – energy production and utilization
•
informational- language for communication inside and between cells
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Practical applications of biochemistry
1. in medicine and health care :
•
enzymes as markers for disease eg lactate dehydrogenase (heart attack
can diagnosed by an increase of ldh from heart muscle
•
acetylcholinesterase (ace) important in controlling certain nerve impulse.
many pesticides interfere with this enzyme.
•
designer drugs – new and improved antibiotics and chemotherapy agents
•
human proteins through genetic recombinant techniques eg insulin, hgh
2. in agriculture – herbicides & pesticides, genetic engineering
3. chemical industries – synthesis & detoxification
Biochemical connections
•
lactic acid and sports
•
neurophysiology – some aa are key precursors to hormones and
neurotransmitters
•
nutrition- aspartame (sweetener), lactose intolerance
•
allied health- phenylketonuria, multiple sclerosis, lupus (autoimmune
disease /immune system attacks the body own tissues involve rna
processing), dental plaque,anemia, atherosclerosis
•
forensic- uses of DNA testing
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CHAPTER 2 : Water, acid and base, buffer
Water
•
essential for life
•
major constituent of almost all life forms
•
most animals and plants contains more than 60% water by volume
•
structure consists of 2H atoms bonded to 1 O atom.
•
the H side of the molecule has a slight +ve and the O side a –ve charge
exist
•
makes it polar and has strong solvent properties
•
hydrophilic compounds interact (disslove) with water eg . polar cpds
(alcohols and ketones)& ionic cpds (kcl), amino acids
•
hydrophobic compounds do not interact with water eg. non polar cpds
(hexane, fatty acids, cholesterol)
Roles of water in the life of organisms
•
mammalian cells 70% water
•
solvent for biological systems & for most chemical reactions that support
life.
•
75% of the earth is covered with water
•
has a very high specific heat-retains heat better than other materials
Some uses of water as solvent
•
flavoring and co2 gas dissolved in water to make soft drinks
•
farmers use water to dissolve fertilizers
•
medicines in water
•
chlorines or flourides added to water
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STUDY GUIDE for BIOCHEMISTRY
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Hydrogen bonds
•
water molecules are hydrogen bonded
•
the ability to form strong h bond is responsible for the many unique
characteristics of water such as its high melting point and boilng point
•
3d structures of many important biomolecules including proteins (Hb) and
nucleic acids (DNA) are stabilized by H bonds
Acids, bases, and buffers
Principle of ionization of weak acids:
•
the fundamental concept of buffers is: a buffer resists change
•
pH buffers resist change in ph when either acid (h+) or base (oh-) is added
to it.
•
chemicals which are ph buffers are weak acids or bases
•
acids = proton (H+) donors
•
bases = proton acceptors
This tendency to ionize can be put in terms of an equation for the
equilibrium:
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STUDY GUIDE for BIOCHEMISTRY
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where [ ] = molar concentration; k = ionization constant (acid dissociation
constant)
Simplest example is water (H2O):
but since [H2O] (water concentration) = constant (55.5 m), kw = [h+][oh-] = 10-14
M
in pure water, [h+] = [oh-] = 10-7 m
•
to make this easier to use, the ph scale was invented.
•
pH = -log [h+]; thus when [h+] = 10-7 m, ph = 7
•
this is called neutral ph because it is in the middle of the ph scale. at ph
greater than neutral, the solution is alkaline; while at ph less than neutral,
the solution is acid.
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TITRATION OF A WEAK ACID ILLUSTRATING ITS IONIZATION AND
BUFFERING PROPERTY
•
all weak acids have titration curves like this one. bases (like ammonium,
nh4+) are also weak acids and have similar titration curves.
•
the position where the buffering zone is on the ph scale is related to the
chemical nature of the weak acid:
•
acetic acid ionizes in the acidic portion of the pH scale
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STUDY GUIDE for BIOCHEMISTRY
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•
this relationship is known as the Henderson-Hasselbalch equation.
•
useful in predicting the properties of buffer solutions used to control the pH
of reaction mixtures.
•
the pk of a weak acid is the ph where [a-] = [ha]
•
at pH below the pk, [HA] > [A-]
•
at pH above the pk, [HA] < [A-]
•
therefore the pk determines the buffering zone for a weak acid.
•
a similar expression pk can be used, pk=-log k
•
the ph of a solution of a weak acid and its conjugate base is related to the
concentration of the acid and base- Henderson- Hasselbach equation.
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2009
for example, acetic acid has a pk = 4.8 and a buffering zone from ph 3.8 to
5.8.
•
so a weak acid will be an effective buffer at ph = pk +/- 1 ph unit.
summary
•
acids are proton donors and base are proton acceptors
•
water can accept or donate protons
•
the strength of an acid is measured by its acid dissociation constant, k
•
the larger the k value, the stronger the acid and more h+ dissociates
•
the conc. h+ is expressed as ph, -ve log of H ion conc.
Calculating pH for weak acids and bases
Calculate the relative amounts of acetic acid and acetate ion present at the
following points when 1 mol of acetic acid is titrated with NaoH. use HH eqn. to
calculate ph
1. 0.1 mol NaOH added
2. 0.3 mol NaOH added
3. 0.5 mol NaOH added
ratio 1:1, when 0. 1 mol of naoh added, 0.1 mol acetic acid reacts with it to form
0.1 mol acetate ion, leaving 0.9 mol acetic acid
ph = pk + log 0.1/0.9
= 4.76 -0.95
= 3.81
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TUTORIAL 1, ENV 416/400
1. Calculate the hydrogen ion concentration for each of the following materials:
a)
b)
c)
d)
e)
Blood plasma, pH 7.4
Orange juice, pH 3.5
Human urine, pH 6.2
Household ammonia, ph 11.5
Gastric juice, pH 1.8
2. Define the following :
a)
b)
c)
d)
e)
f)
Acid dissociation constant
Equivalence point
Hydrophilic
Hydrophobic
Non polar
Polar
3. What is the [CH3COO-] / CH3COOH ratio in an acetate buffer at pH 5.00?
4. What are some macromolecules that have hydrogen bonds as part of their
structures?
5. What is the relationship between pKa and the useful range of a buffer?
6 What is the pK of a weak acid HA if a solution containing 0.2M HA and 0.1M
A- has a pH 0f 6.5 ?
7. Explain buffer solution. Give an example of a buffer solution.
8. Explain why polar substances dissolve in water while non polar substances do not.
9. Explain why a 1M solution of HCl has a pH of 0.
10. A 5.0 ml of H2SO4 is titrated with 0.2 M KOH to neutrality. If 4.5 ml of KOH
was used what was the pH of the original acid?
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CHAPTER 3 : Amino acids and Proteins
Amino acids and peptides
1. Only 20 aa usually found in proteins
2. The general structure includes an amino group and a carboxyl gp.
3. The α-carbon is bonded to a H and side chain gp (R)
4. The R gp determines the identity of the particular amino acid
Types of Amino Acids based on side-chain chemical character:
I. Non-Polar or hydrophobic (water hating)
II. Flexible
III. Polar or hydrophilic (water loving)
There are 20 Amino Acids encoded by codons in the genetic code:
When there are more than 100 AAs found in nature, why only 20 AAs in proteins?
Because these 20 AAs provide all the chemical and size groups needed to make a very
large number of proteins. Plus many of these amino acids become modified after
translation into proteins, which increases the available chemical character of amino acid
side chains.
These 20 AAs can be divided into the above 3 groups (non-polar, flexible and polar) and
then subdivided by their chemical character:
Group I = Non-Polar -- 8 AAs
Hydrocarbon NON-POLAR AMINO ACIDS -- 5 AAs -- Ala Val Leu Ile Pro:
Non-Polar -- Hydrocarbon -- Ala (Alanine)
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STUDY GUIDE for BIOCHEMISTRY
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The chiral Carbon of Ala is emphasized here! All amino acids are derivatives of Ala,
except Gly
Non-Polar -- Hydrocarbon -- Val (Valine)
Val has to methyl groups added to Ala to make an isopropyl group.
Non-Polar -- Hydrocarbon -- Leu (Leucine)
Leu adds an isopropyl group to Ala so that Leu has 4 carbons in its side chain.
Non-Polar -- Hydrocarbon -- Ile (Isoleucine)
Ile is a structural isomer of Leu so it also has 4 carbons in its side chain. But Ile is bulkier
than Leu near the base of the side chain, while Leu is bulkier than Ile farther out on the
side chain (size/shape of side chains is important). Ile has a 2nd chiral center which is
emphasized in the Ile drawing above.
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Non-Polar -- Hydrocarbon -- Pro (Proline)
Pro is a very special amino acid due to its inflexible character!!! Pro is inflexible because
its side chain bonds to alpha-amino group in a ring structure which can not twist around
the bond between alpha-amino group and alpha carbon, which all other AAs can. Also
Pro, thus, has a secondary amino group (notice the single hydrogen on its Nitrogen atom)
with different chemical character than the primary amino groups in all other amino acids,
which have two hydrogens on them.
Aromatic NON-POLAR AMINO ACIDS -- 2 AAs --Phe
Trp:
Non-Polar -- Aromatic -- Phe (Phenylalanine)
Phe adds a benzene ring to Ala!
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Non-Polar -- Aromatic -- Trp (Tryptophan)
Trp has a heterocyclic aromatic group with an aromatic amine in it.
Thiol Ether NON-POLAR AMINO ACID -- 1 AA -- Met:
Non-Polar -- Thiol Ether -- Met (Methionine)
Met introduces the important Sulfur element into proteins which is found in Cys also (see
below). Met contains a thiol ether (R-S-R) in its side chain, which is much less polar than
an oxy-ether (R-O-R) like the compound we call ether, which is an polar organic solvent.
Met is a very hydrophobic AA.
Group II = Flexible -- 1 AA -- Glycine is the Flexible Amino
Acid
Flexible -- Gly (Glycine)
Gly is a unique AA with no chiral center -- but it is prochiral since it has two groups the
same (ie H) on the central carbon -- so it still has sidedness - try making a model of Gly.
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Most important since Gly has no side chain it is very flexible and can easily twist around
its alpha-amino Nitrogen bond to the alpha-Carbon. Gly is the opposite of Pro - Gly is
flexible while Pro is inflexible.
Finally, Gly makes a transition from the non-polar AAs to the polar AAs. Gly is neither
nonpolar or polar .
Group III = Polar -- 11 AAs
THE POLAR AMINO ACIDS
Polar AAs are important since they provide chemical groups for interaction with water.
Thus, the hydrogen bonding character of polar AAs is key in forming protein structures.
While the ionic bonding character of charged polar AAs is also important in protein
structure. Also the polar side chains in these AAs provide the chemically reactive groups
in proteins.
Alcohols - Neutral Polar Amino Acids -- 3 AAs -- Ser Thr & Tyr:
Polar -- Neutral -- Alcohols -- Ser (Serine)
Ser contains one -OH group and so it is essentially hydroxy-Ala. The hydroxyl group on
Ser does not normally ionize, so it is not charged in proteins - its neutral. Ser is the
smallest AA of the polar amino acids and is very polar. The hydroxyl group on Ser
provides enzymes a very good nucleophilic group for doing chemistry. Another important
function of Ser is to form esters with phosphate, making phospho-ester proteins.
Phosphorylation of proteins/enzymes is very important in regulation of activity.
Polar -- Neutral -- Alcohols -- Thr (Threonine)
Thr adds a Carbon on to Ser, which makes the hydroxyl group less accessible in Thr than
Ser. Thr serves more often in a structural role in proteins and is usually not as chemically
active as Ser. Thr can form esters with phosphoric acid and phospho-Thr is often found in
proteins.
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Polar -- Neutral -- Alcohols -- Tyr (Tyrosine)
Tyr is an aromatic alcohol and so it has both aromatic character and polar character. The
hydroxyl of Tyr is like the hydroxyl in phenol, so at high pH it can ionize. Tyr can also
form phospho-esters like Ser and Thr. Phospho-Tyr is very important in proteins/enzymes
involved in regulating the cycle cell.
Thiol - Neutral Polar Amino Acid -- 1 AA -- Cys:
Polar -- Neutral -- Thiol -- Cys (Cysteine)
Cys is essentially thiol-Ala. The thiol (-SH) group of Cys can ionize as shown in graphic.
Thiols ionize at about pH 8 and so usually they are protonated at biological pH. Hydroxyl
groups like in Ser have pK about 15 or so and do not ionize normally.
A Special Feature of Cys is that it can oxidize (in the presence of oxygen) and
react with another Cys to form Cystine or a disulfide bond:
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The formation of "Cystine" can take place between 2 polypeptide chains to make a crosslink between them. This is actually an enzyme catalyzed reaction which takes place in the
lumen of ER in cells when proteins are being exported from the cell. A very good
example is the production of antibodies by cells in the immune response - antibody
proteins contain many Cys- Cys or disulfide bonds. Excellular proteins often contain CysCys bonds, while cellular proteins do not usually contain the Cys-Cys since the conditions
in the cell are reducing. In the second part of the graphic above, the general reaction of 2
thiols is shown. In the presence of oxygen or oxidizing conditions, the 2 thiols react to
form a disulfide bond between them. Since this is a redox reaction, the hydride ion
released by each thiol is usually coupled to an electron acceptor reaction or in simple
oxidiation with oxygen, hydrogen peroxide is usually formed with further reduction to
water.
Amides - Neutral Polar Amino Acids -- 2 AAs -- Asn & Gln:
Polar -- Neutral -- Amides -- Asn (Asparagine)
Asn is a very small amino acid as well as being very polar. Amides are neutral and do not
ionize nor do they accept protons.
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Polar -- Neutral -- Amides -- Gln (Glutamine)
Gln is a bit larger amide than Asn because it has a longer side chain string of Carbons.
Both the amide AAs are neutral derivatives of the corresponding acid AAs (Asp & Glu see below) Understanding the chemical character of the amide is very important, since the
peptide bond of proteins is an amide bond.
Acids - Negatively Charged Amino Acids -- 2 AAs -- Asp & Glu:
Polar -- Charged -- Acids -- Asp (Aspartic acid or Aspartate)
Asp has a second carboxylic acid group in addition to its alpha-carboxylic acid group. The
Asp side chain carboxyl group is normally ionized at biological pH; Asp a negatively
charged AA. Asp is a rather small AA and is very polar.
Polar -- Charged -- Acids -- Glu (Glutamic acid or glutamate)
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Glu also has a second carboxylic acid group in addition to its alpha-carboxylic acid group.
The Glu side chain carboxyl group is normally ionized at biological pH; Glu is negatively
charged.
Bases - Positively Charged Amino Acids -- 3 AAs -- Lys Arg & His:
Polar -- Charged -- Bases -- Lys (Lysine)
Lys has a primary amino group at the end of a 4 Carbon side chain and it can be positively
charged. Since the Lys side chain amino group has a high pK , it is often charged
at biological pH.
Polar -- Charged -- Bases -- Arg (Arginine)
Arg has a complex side chain containing 3 Nitrogen groups, which work as a unit to give
a positive charge. Since the Arg side chain group has a very high pK , it is always
charged at biological pH. Arg provides proteins/enzymes with essentially a fixed positive
charge.
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Polar -- Charged -- Bases -- His (Histidine)
His has an aromatic-like pair of amino groups, making His a unique AA with a positive
charge -- sometimes. His with a pK for its side chain near neutrality, means that it can
either be charged or not at biological pH. His, when not charged, is a very strong
nucleophile and is very important in enzyme chemistry. His is also very important as a
proton acceptor and donor in biochemical
reactions.
Protein Covalent Structure (Protein Primary Structure)
I. Peptide Bonds, Peptides and Proteins
Proteins are sometimes called Polypeptides, since they contain many Peptide Bonds
The peptide bond is an amide bond
Water is lost in forming an amide bond.
Structural Character of Amide Groups: Understanding the chemical character of the
amide is very important, since the peptide bond of proteins is an amide bond.
Amides have a partial double bond character and also a partial charge character because
of the resonance forms shown in the above graphic.
Comparison of an amino acid, a dipeptide and a tripeptide:
Amino Acid = Gly; dipeptide = Gly-Ala; tripeptide = Gly-Ala-Ser
Peptides = Mini-Proteins
A pentapeptide -- GlyAlaSerPheGln
1st amino acid is always written on the left and called the Amino terminal, since it is
always the only amino acid of the peptide with a free alpha-amino group. Last amino acid
is always written on the right and called the Carboxyl terminus, since it is always the only
amino acid of the peptide with a free alpha-carboxylic acid group.
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List of Proteins Shown in Amino Acid Composition Table:
A. Antibody - Human Bence-Jones Kappa (antibody light chain)
B. Human Cytochrome c (electron transport protein)
C. Spinach Ferredoxin (electron transport protein)
D. Pig Glucagon (protein hormone)
E. Bovine Insulin (protein hormone)
F. Human/Gorilla Hemoglobin alpha chain (oxygen transport protein)
G. Human/Gorilla Hemoglobin beta chain (oxygen transport protein)
H. Chicken Lysozyme (enzyme)
I. Sheep Wool (structural protein)
Free amino acids are obtained from proteins by strong acid hydrolysis:
B. OVERALL CONFORMATION OF PROTEINS
Proteins have a covalently bonded backbone as discussed in Lecture 5 in relation to amino
acid sequence determination. But the 3-D shape or conformation is held together by
weaker bonding of the non-covalent type. The linear form of the polypeptide backbone of
the protein folds into a tightly held shape which is chemically stabilized by weak bonds
like hydrogen bonds, ionic bonds and hydrophobic interactions among non-polar amino
acid side chains.
To reduce the complexity of protein structure to a manageable level for our study and
understanding, the protein is considered to have 4 levels of structure.
Four Levels of Protein Structure:
1. Primary Structure- Polypeptide backbone
2. Secondary Structure- Local Hydrogen bonds along the backbone
3. Tertiary structure- Long distance bonding involving the AA side chains
4. Quaternary structure- Protein-Protein interactions leading to formation of dimers,
tetramers, etc.
C. PRIMARY STRUCTURE OF PROTEINS
We have already discussed the Primary structure of Proteins, which is the polypeptide
backbone or amino acid sequence. The amide bonds joining the individual amino acid
residues of the backbone have an important role in forming the 3-D structure of proteins.
The peptide bond of the amino acid sequence forms a planar structure due to the partial
double bond between N and C. This planar structure limits the ways the backbone can
fold up and therefore, constrains the shape a folded polypeptide can take.
The Amide Bond showing its partial double bond character and partial charges.
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D. SECONDARY STRUCTURE OF PROTEINS
In 1950's, Linus Pauling named the first structures he found by X-ray diffraction, the
Alpha Helix and the second structure he found was called Beta Sheet. We continue to use
these names today for two forms of secondary structure and add a third type forms in
regions where the protein bends back on itself to form its compact shape or conformation.
The 3 Types of Protein Secondary Structure:
Alpha-helix
Beta-sheet
Turns or Bends (Bends in backbone to fold the polypeptide back on itself)
E. LOCAL HYDROGEN BONDING FORMS SECONDARY STRUCTURE
Secondary Structure is formed by local Hydrogen Bonding between the Hydrogen on the
Nitrogen of one amide in a peptide bond with carbonyl oxygen of another amide in a
second peptide bond.
Hydrogen bonds (H-bonds)are weak non-covalent bonds. The energy required to break an
Hbond is about 1 to 4 kcal/mole as compared to a covalent bond which requires about 100
kcal/mole to break. Thus, H-bonds are a bit flexible and for example, the H-bonds holding
water together as liquid constantly break and reform. However, in more directed H-bonds
like found in protein secondary structure, the pair of groups involved stay as partners and
with the overall arrangement of a single H-bond being stabilized by a group of H-bonds.
So H-bonding in secondary structure is stronger due to the local grouping of these bonds
and secondary structure forms like the alpha-helix and beta-sheet are neighborhoods of Hbonds acting together like a group.
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Figure 7. Hydrogen Bond (H-Bond) between Two Peptide Bonds.
.
Model of Right-Handed Alpha-Helix Showing H-Bonding (From Voet/Biochemistry
1990 John Wiley)
Model of Beta Sheet Showing H-Bonding between Two Strands of the Sheet. (From
Voet/Biochemistry ©1990 John Wiley)
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F. Alpha-HELIX
Alpha helix is held together by hydrogen bonds between the amide Hydrogen on the
Nitrogen and another amide carbonyl oxygen of every 4th amino acid residue
(approximately). These are intrachain H-bonds which along the same region of the
backbone of the polypeptide or in other words within the same region of the amino acid
sequence.
Simple Model of Alpha Helix with H-bonding Pattern.
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The side chains of the amino acids project out from the core of the alpha helix. Water is
excluded from the tight inner core of the alpha helix, which is very hydrophobic.
G. Beta SHEET
Beta sheets are also held together by hydrogen bonds between the Hydrogen on the
Nitrogen and another amide carbonyl oxygen of the peptide bonds but between chains of
the backbone rather than along it as was found for the Alpha helix. These are called
interchain H-bonds since they form between two parts of the polypeptide backbone
separated from one another by some distance or length of the amino acid sequence of the
polypeptide.
Simple model of H-Bonding in a Beta Sheet.
Two types of backbone chain order is found:
1. PARALLEL where the chains run in the same direction
2. ANTI-PARALLEL where chains run in the opposite direction
Models of (a) Antiparallel and (b) Parallel Beta Sheets (Only two strands of beta-sheet
shown).(From Voet/Biochemistry ©1990 John Wiley)
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H. TURNS AND BENDS IN THE POLYPEPTIDE BACKBONE
Proline (Pro) breaks up secondary structures like alpha-helix and beta-sheet. Because Pro
can not bend, Pro is often found at the ends of Alpha Helix and Beta Sheet strands. Thus,
the third type of Secondary Structure is actually formed by the absence of the other two
types.
Positions of Pro in Relation to Alpha-Helix and Beta Sheet Secondary Structures
Places where the polypeptide backbone bends so that the protein can fold back on itself to
form the compact structure also have hydrogen bonds in some cases. These H-bonds
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occur only between the 1st and 4th amino acid residue of the Reverse Turn and no other
H-bonds are formed.
TUTORIAL 2, ENV 416
1. Draw the dipeptide Asp-His
2.
Identify the nonpolar amino acids and the acidic amino acids in the following
peptide :
Glu-Thr-Val-Asp-Ile-Ser-Ala
3. Sketch a titration curve for alanine and indicate the pKa values for all the
titratable groups. Also indicate the pH at which this amino acid has no net charge.
4. Draw 2 hydrogen bonds, one is part of a secondary structure and another that is
part of a tertiary structure.
5. Draw a disulfide bridge between two cysteines in a polypeptide chain.
6. What is the highest level of oragnization in myoglobin and hemoglobin?
7. Differentiate between secondary and tertiary proteins. Name an example for each.
8. Differentiate between alpha-helix and beta sheet.
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CHAPTER 4 : PROPERTIES OF ENZYMES
Enzymes are biological catalysts. Like all catalysts, enzymes lower the energy needed to
get a reaction started. Enzymes are much generally better at accelerating the rates of
reactions than non-biological catalysts.
Figure 1. Diagram showing that less energy is required to get an enzyme catalyzed
reaction started as compared to a non-catalyzed reaction. Figure from Zubay et al.,
Principles of Biochemsitry copyright 1995 Brown Comm.
Enzymes have been divided into 6 classes by the International Commission on Enzyme
Nomenclature. All enzymes are assigned a number (called an EC number) which defines
exactly the reaction catalyzed by the enzyme. For example, trypsin is EC 3.4.21.4 since it
is in class 3 (hydrolases) which work on peptide bonds (3.4) in the middle of proteins
(3.4.21 are serine endopeptidases) - trypsin is the 4th entry in this subclass.
These six classes are:
1. Oxidoreductases - enzymes catalyzing oxidation reduction reactions.
2. Transferases - enzymes catalyzing transfer of functional groups.
3. Hydrolases - enzymes catalyzing hydrolysis reactions.
4. Lyases - enzymes catalyzing group elimination reactions to form double bonds.
5. Isomerases - enzymes catalyzing isomerizations (bond rearrangements).
6. Ligases - enzymes catalyzing bond formation reactions couples with ATP hydrolysis.
These 6 enzyme classes can also be illustrated by the general reactions catalyzed:
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Figure 2. Model reactions of the 6 classes of enzymes. Figure from Zubay et al.,
Principles of Biochemsitry copyright ©1995 Brown Comm.
Examples of enzymes in each class:
1. Alcohol dehydrogenase (EC 1.1.1.1)
2. Hexokinase (EC 2.7.1.1)
3. Trypsin (EC 3.4.21.4)
4. Ribulose-bisphosphate carboxylase (EC 4.1.1.39)
5. Triose phosphate isomerase (EC 5.3.1.1)
6. Tyrosine tRNA ligase (6.1.1.1)
Enzyme Additives (Cofactors) Assisting in Catalysis
Enzymes are often composed of only protein. In this case only AA side chains are used
for catalysis. Some enzymes require additives for assisting with catalysis. Additives like
vitamins often provide functional groups not available to the enzyme among the side
chains of the amino acids.
In these cases the protein of the enzyme binds:
Organic cofactors (Vitamins = organic cofactors)
Metal ions (e.g. Mg2+)
Nucleotides (even RNA)
The Common Cofactors (Enzyme Additives):
Biotin aids in carboxylation reactions (carbon dioxide fixation).
Cobaltamine (vitamin B-12) aids in alkylation reactions (methylation for instance).
Coenzyme A aids in acyl transfers like in the tricarboxylic acid cycle.
Flavin (vitamin B-2) aids in oxidation-reduction reactions (e.g. nitrate reductase).
Lipoic acid aids in acyl transfers via oxidation-reduction processes.
Nicotinamide coenzymes like NAD+ act as independent co-substrates.
Pyridoxal (vitamin B-6) aids in amino group transfers (provides aldehyde functional
group).
Tetrahydrofolate aids in one-carbon transfers.
Thiamin (vitamin B-1) aids in aldehyde transfers and alpha-keto-acids decarboxylations
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The complex of protein and additive is called Holo-Enzyme. When the additive is
removed from the enzyme, the remaining protein part of the enzyme is called the ApoEnzyme.
Apo-Enzyme (inactive) + Additive = Holo-Enzyme (active)
The Active Site of the Enzyme.
Each enzyme has a unique active site.
Active site = catalytic site.
The enzyme binds its substrate(s) at the active site and the enzyme catalyzes chemical
changes in the substrate(s). The types of chemical reactions catalyzed were illustrated
above in
Figure 3. NAD+ bound in the active site of GAP dehydrogenase. The NAD+ molecule is
shown in bold and the side chains of the amino acids binding it are shown projecting from
the surface of the enzyme (shown as the filled in area surrounding the active site).
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Enzymes contain a large number of amino acids, but most AA side chains are used for
forming the enzyme's shape. Only a few AA side chains are at the active site. These
special AA side chains:
1. Bind the substrate(s) and
2. Catalyze the reaction
This concept is illustrated in the following figures by 3 different drawings of the enzyme
ribonuclease which catalyzes the hydrolysis of RNA. The first view is of the 3-D shape of
the enzyme with the 3 key amino acids at the active site highlighted (His12, Lys41 and
His119 - numbers indicating the position of these residues in the amino acid sequence of
ribonuclease).
Next is a ribbon model with the 3 key amino acids shown in relation to the various
secondary structure elements of ribonuclease. Last is a ball-and-stick model of
ribonuclease with the same 3 amino acid side chains of the active site emphasized. A
feature to try to see in these models is the groove of the enzyme which forms the active
site and how the enzyme folds to bring these 3 key amino acid side chains together to
form the active site.
Figure 5. 3-D model of the enzyme ribonuclease with the key amino acid side chains at
the active site shown in red. The active site is a deep groove at the center of this structure.
Summary of the Active Site of Enzymes:
Enzyme has large structure with hundreds of AA side chains but only a few are involved
in catalysis.
Each enzyme has a unique active site.
Key AA side chains are involved in binding and catalysis in the active site.
Enzyme Framework - Why are Enzymes so Large?
We have discussed the formation of a protein's 3-D shape recall- the 4 levels of protein
structure: Primary, Secondary, Tertiary, Quaternary. They make up a "Framework" to
bring the AA side chains of the active site together. By bringing the AA side chains of the
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active site together they can act synergistically or in concert which is part of what makes
enzymes very effective catalysts.
Figure 6. Ribonuclease with substrate RNA model bound in active site. His12 and His119
are involved in catalysis of the phosphodiester bond in the backbone of the RNA. Lys41
assists with binding the RNA molecule.
The active AA side chains also provide the enzyme with a high degree of specificity so
that only certain substrates are bound to the enzyme's active site..
How do enzymes catalyze a reaction???
One answer is: Like all catalysts, enzymes decrease the energy required to get a reaction
started. This was illustrated in the first part of this lecture with an energy diagram. Below
is shown a similar diagram with more detail for the energy pattern for the enzyme
catalyzed reaction. First, energy is required to form the complex between the enzyme and
substrate (E-S complex) which is a higher energy state than the free enzyme and
substrate/product.
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Figure 7. Diagram of energetics of enzyme catalyzed reaction versus non-catalyzed
reaction.
Summary of Enzyme Catalysis:
Enzymes bind substrate with great specificity
Enzyme catalyzed reactions usually have no side products
Enzymes use energy released when substrates bind to make their catalysis more
effective.
Introduction to Enzyme Kinetics.
In chemistry, kinetics has to do with the rate of reactions. In biochemistry, we are most
interested in rates of enzyme catalyzed reactions since virtually all biological reactions are
catalyzed by enzymes.
Enzyme Kinetics: Rates of enzyme catalyzed reactions
Usefulness of enzyme kinetics:
Common clinical assays to detect enzymes
Understanding metabolic pathways
Measuring binding of substrates and inhibitors to the active site of an enzyme
Understanding the mechanism of catalysis of an enzyme
Rates of reactions are measured by change in reactant amounts with time. You can
measure the disappearance of the substrate or the appearance of the product. Usually, the
appearance of the product is easier to keep track of since there should be no product
present at the beginning of the reaction.
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Figure 8. Ways to express a rate for the enzyme catalyzed reaction.
Rates = Reaction Velocity
For enzymes, the initial velocity (before significant product accumulates) is always used.
Initial Velocity = Vo
A Simple Mechanism for the Enzyme Catalyzed Reaction.
For catalysis to begin, the substrate must bind to the enzyme, which results in the
formation of the enzyme-substrate complex (ie E-S complex). The E-S complex forms
rapidly in the first part of the enzyme catalysis process and the concentration of the E-S
stays constant at a steady-state level. For this reason, this type of kinetics is called steadystate kinetics.
A simple mechanism for the enzyme catalyzed reaction helps us to understand and model
this process.
E + S ↔ ES → E + P
A simple enzyme mechanism for a single substrate and product.
Enzyme Catalyzed Rates at Different Substrate Concentrations.
Since the enzyme is used many times to catalyze the same reaction, the concentration of
the enzyme is much less than the substrate:
[S] >> [E]
Thus, the substrate saturates the enzyme. This is best understood by observing the rate of
the reaction or initial velocity at different [S] (ie. substrate concentrations):
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[S] mM
0
1
2
5
10
50
100
2009
Vo μmol product/min
0.0
0.9
1.4
1.9
2.3
2.6
2.6
Model data for the enzyme catalyzed reaction. These data show that at low [S], the initial
velocity is more or less proportional to the [S]. At high [S], the initial velocity no longer
increases as more substrate is added. Thus, at high [S] the enzyme is saturated with
substrate and no increase in the enzyme catalyzed rate is observed.
This model set of data for an enzyme catalyzed reaction shows the initial velocity in terms
of the amount of product formed per unit time (ie micromoles of product produced/min) at
various substrate concentrations. These data can be plotted in a graphical form to also
illustrate the results of an enzyme catalyzed reaction.
Plot of initial velocity of the enzyme catalyzed reaction (Vo) versus the [S] (ie
substrate concentration). Initial velocity is always given in units of amount of product
formed per unit time and the substrate concentration is given in molar units (ie mM).
Here it is easy to see the saturation of the enzyme at high [S] where the initial velocity
approaches a limiting value. The plot has the shape of a square hyperbola.
The Michaelis-Menten Equation.
The plot of Vo versus [S] can be represented by an equation, which is known as the
Michaelis- Menten equation in honor of the scientist who first described it. This equation,
sometimes called the M-M equation, is an important one for you to know and understand.
v0 = Vmax [S ]
Km + [S ]
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The Michaelis-Menten equation which describes the change in Vo as [S] increases.
The constants in this equation, Km and Vmax, are defined:
Vmax = Maximum velocity catalyzed by a fixed [E]
Km = the [S] which gives 1/2 Vmax
These definitions are illustrated below:
Vo versus [S] plot illustrating the operational definitions of Vmax and Km.
Thus, the limit approached in the Vo versus [S] plot is the Vmax.
Definition of Km and Vmax and Their Ratio - Vmax/Km.
The Km is sometimes called the Michaelis Constant. The Km is an intrinsic property of an
enzyme related to the binding constant for forming the ES complex, which is an
equilibrium and can be defined by the rate constants for its formation and breakdown
using the simple enzyme mechanism shown above.
The approximate relationship between the Km and the Ks for the binding of the
substrate to the enzyme which leads to the formation of the E-S complex. Ks is defined by
the equilibrium formed between the enzyme (E) and substrate (S) and the E-S complex, as
shown above. Ks is also defined by the ratio of the rate of breakdown of the E-S complex
divided by its rate of formation.
But Km also involves the breakdown of the E-S complex to E and P, which is not a
component of the Ks. Thus, the rate of the breakdown of the E-S complex to make
product (P) is also defined in the simple enzyme mechanism .
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The definition of Km by using rate constants for simple enzyme mechanism. The point
of this graphic is to emphasize that the Km constant of the enzyme catalyzed reaction
includes more than just the formation of the E-S complex, but also its breakdown to form
product, which is of course the key to an enzyme catalyzed reaction.
So Km reflects both binding of E to S but also the catalytic constant (shown as k3 above,
but also defined as kcat) of the enzyme catalyzed reaction.
The Vmax is also dependent on the catalytic constant:
Vmax = kcat [E]
So both Vmax and Km are properties of individual enzymes and not very useful for
comparing enzymes.
However, the ratio Vmax/Km can be used to compare enzymes. This ratio (Vmax/Km)
measures the efficiency of the enzyme. The efficiency of the enzyme is ultimately limited
by the rate of diffusion of the substrate to the enzyme - thus the diffusion of substrates,
which is very rapid, sets an upper limit. The most efficient enzymes like Triose-P
Isomerase are limited by how fast their substrates get to them. But most enzymes are not
this efficient and more limited by chemical events in the active site of the enzyme.
Finding the Km and Vmax by the Graphical Solution Method.
To calculate the Km and Vmax, the Michaelis-Menten equation is converted into a linear
form by taking the reciprocal of both sides of the equation. This is called the LineweaverBurk equation in honor of the first scientists to describe it.
The Lineweaver-Burk equation linearizes the M-M equation by taking the reciprocal of
both sides of the equation. This equation then takes on the form of the equation of a line.
The y values are 1/Vo, the x values are 1/[S]. The b value in the line equation is the slope
and equal to Km/Vmax, while the c value is the y-intercept and equal to 1/Vmax.
The double reciprocal plot is useful for deriving Km and Vmax by plotting kinetic data for
an enzyme and you should use it to find the Km and Vmax via graphing for the problem
set you got today.
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The double reciprocal plot for enzyme kinetic data.
This plot must be used to find Km and Vmax for enzyme kinetic data in this class as
shown on the graphic. The y-intercept is the 1/Vmax. The x-intercept, which is found in
the 4th quadrant, is -1/Km. Alternatively, the Km value can be found from the slope using
the Vmax value found from the y-intercept.
However, there are statistical problems with the Lineweaver-Burk equation and double
reciprocal plots, so today in research, one derives Km and Vmax using other methods
such as the direct linear plot using a computer program. However, the Lineweaver-Burk
equation makes the clearest representation of kinetic data and makes it easy to understand
the results, so it is most often used to illustrate the data even when the Km and Vmax are
derived by other methods.
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Enzyme Inhibitors. A. Competitive Inhibition
Inhibitors of enzymes: Two types are considered - Competitive and Non-Competitive.
A Competitive Inhibitor has a chemical similarity to the substrate and competes with the
substrate for binding to the active site of the enzyme. A good example to describe
competitive inhibition is the mitochondrial enzyme, succinate dehydrogenase:
(A) The reaction catalyzed by succinate dehydrogenase is the oxidation of succinate to
fumarate. (B) Malonate and oxaloacetate are competitive inhibitors of succinate
dehydrogenase.
Both these competitive inhibitors, malonate and oxaloacetate, look like succinate in their
chemical character. Both inhibitors are dicarboxylic acids like the substrate succinate so
they have groups which can bind in the same places in the active site of succinate
dehydrogenase as the substrate. However, neither inhibitor has the capacity to undergo the
reaction and so the enzyme is inhibited. Since these inhibitors simply bind to the enzyme,
when the succinate concentration is high, they will be pushed out of the site by the
substrate and the enzyme will catalyze the reaction as if no inhibitor were present.
An enzyme mechanism model of the action of a competitive inhibitor (Ic) based on the
standard model of a Michaelis-Menten enzyme where E + S leads to the E-S complex,
which leads to product P:
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Model of a Competitive Inhibitor (Ic) Interacting with the Enzyme (E) and an
equation for the equilibrium formed between the Ic and E, which is governed by the
inhibitor binding constant, Ki.
This model is the same as the one described in the previous lecture where enzyme (E) and
substrate (S) bind to form the ES complex, which will go forward during catalysis to form
product (P) and the free enzyme. In the presence of the competitive inhibitor, Ic, a
complex forms with enzyme when the inhibitor binds, the E-Ic complex. This is a deadend complex and can not go on to form product. However, the Ic is bound reversibly to
the enzyme and when more substrate is added, the inhibition is overcome by pulling the
enzyme free via the breakdown of the E-Ic complex, which is in equilibrium with free
enzyme and free Ic. Another way to think about this is - when lots of substrate is added,
the concentration of free enzyme (E) falls to such a low level, that some of the E-Ic
complex must breakdown to replenish the free E demanded by the equilibrium between E
and Ic. This can also be demonstrated by comparing the Vo versus [S] plots for
uninhibited enzyme and enzyme in the presence of a competitive inhibitor:
Vo versus [S] plot comparing the kinetics of the reaction in the absence of inhibitor and in
the presence of the competitive inhibitor (Ic). At high [S], the initial velocity in the
presence of Ic will be about the same as it is in the absence of the inhibitor. The
concentration of S which will be required to overcome the effect of the competitive
inhibitor will depend on the [Ic] (ie. concentration of the competitive inhibitor) and the Ki
(ie. the binding constant of the inhibitor to enzyme).
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In competitive inhibition, addition of more substrate will out compete the inhibitor and
overcome the inhibition of the enzyme's catalytic rate - thus, the Vmax will be the same
and only Km will be altered. This is most clearly illustrated with the double reciprocal
plot comparing the uninhibited reaction to that in the presence of Ic.
Double reciprocal plot for competitive inhibitor (Ic).
Here the uninhibited reaction gives the standard double reciprocal plot from which Km
and Vmax can be calculated. The reaction in the presence of the competitive inhibitor
yields apparent constants for the enzyme which are called the Km' and Vmax'. For the true
competitive inhibitor, the Vmax' (apparent Vmax for inhibited enzyme) will be the same
as the real Vmax, while the Km' (apparent Km for the inhibited enzyme) will be greater
than the real Km. Thus, the -1/Km' will be smaller than -1/Km. After finding Km and
Km', the Ki for the Ic can be calculated using the equation shown using the given
concentration of the competitive inhibitor ([I]).
Enzyme Inhibitors B. Non-competitive Inhibition.
A Non-Competitive Inhibitor does not compete with substrate and the [S] has no
influence on the degree of inhibition of the enzyme's catalytic rate. For example, enzymes
with a thiol ( -SH ) not at the active site can be inhibited:
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Example of a heavy metal inhibiting an enzyme by binding to a thiol group not at the
active site and inactivating the enzyme. Non-Competitive Inhibition can be model using
the standard model for the Michaelis-Menten enzyme where E + S form the ES complex
which leads to formation of product P. In this case where the non-competitive inhibitor
(Inc) reacts with the enzyme at a site other than the active site, both the free enzyme (E)
and the enzyme-substrate complex (E-S) react with Inc. Clearly, in this case the reaction
of the non-competitive inhibitor is irreversible and the substrate can not over come the
inhibitors impact on the enzyme:
Model of the Non-Competitive Inhibitor (Inc). The equilibrium between enzyme and
Inc now depends on the total concentration of enzyme in all forms present (ie. both the
free E and the E-S complex) and defines the Ki.
A Vo versus [S] plot for the Non-competitive Inhibitor looks very different than that for a
competitive inhibitor since increasing the [S] has no impact:
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Vo versus [S] plot for enzyme in the absence and presence of Inc.
The double reciprocal plot for this same model shows that Inc decreases Vmax, as if some
of the enzyme had been removed from the system. In classic example of pure noncompetitive inhibition, the uninhibited reaction and the enzyme in the presence of Inc will
yield the same Km value.
Double Reciprocal plot for the Non-Competitive Inhibitor (Inc).
Non competitive inhibitors decrease Vmax but have no effect on Km.
The apparent Vmax' is smaller than the real Vmax and the Ki for the Non-Competitive
Inhibitor can be calculated using the following equation and the known [I]:
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Equation showing the relationship between Vmax' (apparent Vmax) and real Vmax in
the presence of a Non-Competitive Inhibitor. Use this equation for calculating the Ki of
the Non- Competitive Inhibitor at known [Inc].
Evaluating Enzyme Inhibitors to determine type and their Ki.
To determine what type an inhibitor is:
1. Find Km and Vmax for uninhibited from 1/Vo vs 1/[S] plot.
2. On same graph find Km' and Vmax' for inhibited reaction.
A. If Vmax = Vmax' then inhibitor is competitive type.
(Vmax and Vmax' should not be more than 10% different)
B. If Vmax does not equal Vmax', then if Km = Km', inhibitor is non competitive type.
After finding inhibitor type, then use equations to calculate Ki. Ki is a binding constant
for inhibitor to the enzyme. Ki has same units as the [I]. If [I] = mM, then Ki = mM.
Equations used for calculating Ki values:
Equation for Competitive Inhibitor.
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Equation for Non-Competitive Inhibitor.
Rearrange these equations to solve for Ki.
Tutorial 3, ENV 416
1. An enzyme catalyzed reaction has a Km of 1mM and Vmax of 5 nMs-1, What is
the reaction velocity when the substrate concentration is
(a) 0.25 mM
(b) 1.5 mM
2. For an enzymatic reaction, draw a plot to explain how it catalyzes a reaction.
3. (a) Differentiate between competitive inhibitor and non competitive inhibitor .
(b) Which of this is affected by change in the substrate concentration? Why?
4. Calculate Km and Vmax from the following data:
[S] (µM)
v0 (mM s-1)
0.1
0.34
0.2
0.53
0.4
0.74
0.8
0.91
1.6
1.04
5. Write out the enzyme mechanism model of action for competitive and non
competitive inhibitor based on the MM equation.
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CHAPTER 5 : Nitrogen metabolism
Amino Acid Metabolism
Will be interested in two things:
1) origin of nitrogen atoms and their incorporation into amino group
2) origin of carbon skeletons
AMINO ACID SYNTHESIS
Nitrogen fixation
Gaseous nitrogen is chemically unreactive due to strong triple bond.
To reduce nitrogen gas to ammonia takes a strong enzyme --> reaction is called
nitrogen fixation.
Only a few organisms are capable of fixing nitrogen and assembling amino acids
from that.
+
Higher organisms cannot form NH4 from atmospheric N2.
Bacteria and blue-green algae (photosynthetic procaryotes) can because they
possess nitrogenase.
Enzyme has two subunits:
1) strong reductase - has Fe-S cluster that supplies e- to second subunit
2) two re-dox centers, one of which is a nitrogenase
+
Composed of iron and molybdenum that reduces N2 to NH4
Reaction is ATP-dependent, but unstable in the presence of oxygen.
Enzyme is present in Rhizobium, symbiotic bacterium in roots of legumes (i.e.
soybeans)
Nodules are pink inside due to presence of leghemoglobin (legume hemoglobin)
that binds to
oxygen to keep environment around enzyme low in oxygen
(nitrogen fixation requires the absence of oxygen)
Plants and microorganisms can obtain NH3 by reducing nitrate (NO3-) and nitrite
-
(NO2 ) --> used to make amino acids, nucleotides, phospholipids.
Assimilation of Ammonia
Assimilation into amino acids occurs through glutamate and glutamine.
-amino group of glutamate by
transamination.
Glutamine contributes its side-chain nitrogen in other biosynthetic reactions.
Reaction:
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+
NADPH +H NADP
+
NH4
-ketoglutarate
2009
+
glutamate + H2O
glutamate dehydrogenase
Another reaction that occurs in some animals is the incorporation of ammonia into
glutamine via glutamine synthetase:
+
+
glutamate + NH4 + ATP
glutamine + ADP + Pi + H
When ammonium ion is limiting, most of glutamate is made by action of both
enzymes to produce the following (sum of both reactions):
+
NH4
+ Pi
-ketoglutarate + NADPH + ATP
+
glutamate + NADP + ADP
Transamination Reactions
Having assimilated the ammonia, synthesis of nearly all amino acids is done via
tranamination reactions.
Glutamate is a key intermediate in amino acid metabolism
-amino acid.
transaminase
<------------->
-amino acid1
-amino acid2
-keto acid2
-keto acid1
Origins of Carbon Skeletons of the Amino Acids
Amino acids that must be supplied in diet are termed essential; others are
nonessential.
Although the biosynthesis of specific amino acids is diverse, they all share a
common feature - carbon skeletons come from intermediates of glycolysis,
PPP, or citric acid cycle.
There are only six biosynthetic families:
1) Derived from oxaloacetate --> Asp, Asn, Met, Thr, Ile, Lys
2) Drived from pyruvate --> Ala, Val, Leu
3) Derived from ribose 5-phosphate --> His
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4) Derived from PEP and erythrose 4-phosphate --> Phe, Tyr, Trp
5) Derived from a-ketoglutarate --> Glu, Gln, Pro, Arg
6) Derived from 3-phosphoglycerate --> Ser, Cys, Gly
Porphyrin Synthesis
First step in biosynthesis of porphyrins is condensation of glycine and succinyl
-aminolevulinate synthase.
Translation of mRNA of this enzyme is feedback-inhibited by heme
Second step involves co
-aminolevulinate to form
-aminolevulinate dehydrase.
Third step involves condensation of four porphobilinogens to form a linear
tetrapyrrole via porphobilinogen deaminase.
This is cyclized to form uroporphyrinogen III.
Subsequent reactions alter side chains and degree of saturation of porphyrin ring
to form protoporphyrin IX.
Association of iron atom creates heme; iron atom transported in blood by
transferrin.
Inherited or acquired disorders called porphyrias are result of deficiency in an
enzyme in heme biosynthetic pathway.
congenital erythropoietic porphyria - insufficient cosynthase (cyclizes
tetrapyrrole)
Lots of uroporphyrinogen I, a useless isomer are made
RBCs prematurely destroyed
Patient’s urine is red because of excretion of uroporphyrin I
Heme Degradation:
Old RBCs are removed from circulation and degraded by spleen.
Apoprotein part of hemoglobin is hydrolyzed into amino acids.
First step in degradation of heme group is cle
-methene bridge to form
biliverdin, a linear tetrapyrrole; catalyzed by heme oxygenase; methene bridge
released as CO.
Second step involved reduction of central methene bridge to form bilirubin;
catalyzed by biliverdin reductase.
Bilirubin is complexed with serum albumin --> liver --> sugar residues added to
propionate side chains.
2 glucuronates attached to bilirubin are secreted in bile.
Jaundice - yellow pigmentation in sclera of eye and in skin --> excessive bilirubin
levels in blood
Caused by excessive breakdown of RBCs, impaired liver function, mechanical
obstruction of bile duct.
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Common in newborns as fetal hemoglobin is broken down and replaced by
adult hemoglobin.
AMINO ACID CATABOLISM
Excess amino acids (those not used for protein synthesis or synthesis of other
macromolecules) cannot be stored.
Surplus amino acids are used as metabolic fuel.
-amino group is removed; carbon skeleton is converted into major metabolic
intermediate
Amino group converted to urea; carbon skeletons converted into acetyl CoA,
acetoacetyl CoA, pyruvate, or citric acid intermediate.
Fatty acids, ketone bodies, and glucose can be formed from amino acids.
Major site of amino acid degradation is the liver.
-ketoglutarate to form glutamate,
+
which is oxidatively deaminated to yield NH4 (see pathway sheet).
+
Some of NH4 is consumed in biosynthesis of nitrogen compounds; most
+
terrestrial vertebrates convert NH4 into urea, which is then excreted
(considered ureotelic).
+
Terrestrial reptiles and birds convert NH4 into uric acid for excretion (considered
uricotelic).
Aquatic animals excrete NH4+ (considered ammontelic).
+
In terrestrial vertebrates NH4 is converted to urea via urea cycle.
One of nitrogen atoms in urea is transferred from aspartate; other is derived from
+
NH4 ; carbon atom comes from CO2.
UREA CYCLE
There are six steps of the urea cycle:
+
1) Bicarbonate ion, NH4 and 2 ATP necessary to form carbamoyl phosphate
via carbamoyl phosphate synthetase I (found in mitochondrial matrix).
2) Carbamoyl phosphate and ornithine (carrier or carbon and nitrogen atoms;
an amino acid, but not a building block of proteins) combine to form citrulline
via ornithine
transcarbamoylase
3) Citruilline is transported out of mitochondrial matrix in exchange for
ornithine
4) Citruilline condenses with aspartate --> arginosuccinate via an ATPdependent reaction via arginosuccinate synthetase
5) Arginosuccinate cleaved to form fumarate and arginine via arginosuccinate
lyase
fumarate --> malate--> oxaloacetate --> gluconeogenesis
oxaloacetate has four possible fates:
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1) transamination to aspartate
2) conversion into glucose via gluconeogenesis
3) condensation with acetyl CoA to form citrate
4) conversion into pyruvate
6) Two -NH2 groups and terminal carbon of arginine cleaved to form ornithine
and urea via
arginase
Ornithine is transported into mitochondrion to repeat cycle
Overall reaction:
+
CO2 + NH4 + 3 ATP + aspartate + 2 H2O ---> urea + 2 ADP + 2 Pi + AMP +
PPi + fumarate
Inherited defects in urea cycle:
1) Blockage of carbamoyl phosphate synthesis leads to hyperammonemia
(elevated levels of ammonia in blood)
2) argininosuccinase deficiency
Providing surplus of arginine in diet and restricting total protein intake
Nitrogen is excreted in the form of argininosuccinate
3) carbamoyl phosphate synthetase deficiency or ornithine transcarbamoylase
deficiency
Excess nitrogen accumulates in glycine and glutamine; must then get rid of
these amino acids
Done by supplementation with benzoate and phenylacetate (both
substitute for urea in the disposal of nitrogen)
benzoate --> benzoyl CoA --> hippurate
phenylacetate --> phenylacetyl CoA -->
phenylacetylglutamine
Fate of Carbon Skeleton of Amino Acids
Used to form major metabolic intermediates that can be converted into glucose
or oxidized by citric acid cycle.
All 20 amino acids are funneled into seven molecules:
1) pyruvate
2) acetyl CoA
3) acetoacetyl CoA
4) -ketoglutarate
5) succinyl CoA
6) fumarate
7) oxaloacetate
Those that are degraded to acetyl CoA or acetoacetyl Coa are termed
ketogenic because they give rise to ketone bodies.
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Those that are degraded to pyruvate or citric acid cycle intermediates are termed
glucogenic.
Leucine and lysine are only ketogenic --> cannot be converted to glucose
Isoleucine, phenylalanine, tryptophan, tyrosine are both.
All others are glucogenic only.
C3 family (alanine, serine, cysteine) ---> pyruvate
C4 family(aspartate and asparagine) ---> oxaloacetate
C5 family (glutamine, proline, arginine, histidine) ---> glutamate --ketoglutarate
Methionine, isoleucine, valine, threonine --> succinyl CoA
Leucine --> acetyl CoA and acetoacetate
Phenylalanine and tyrosine --> acetoacetate and fumarate
Tryptophan --> pyruvate
-
Regulation of the Urea Cycle
The main allosteric enzyme is glutamate dehydrogenase.
It is inhibited by high GTP and ATP levels.
It is stimulated by high GDP and ADP levels.
Phenylketonuria
Phenylketonuria is (at least among Europeans) the most common hereditary
enzyme defect. It is clinically manifest in about one among ten thousand
persons. Considering that only homozygous people are clinically affected,
this works out to a heterozygote frequency of (4×1/10,000)½ = 1/50,
i.e. one in fifty persons can potentially have children with this disease.
The enzyme affected is phenylalanine hydroxylase, the first enzyme in the
degradative pathway . The name of the disease stems from the fact that
phenylpyruvate and some derivatives thereof are found in the urine.
Formation of phenylpyruvate is due to the buildup of phenylalanine, which
will eventually cause it to overcome the low KM of tyrosine transaminase .
Phenylpyruvate is believed to give rise to neurotoxic metabolites, although
the exact nature of these metabolites remains to be elucidated. Symptoms
include disturbances in neurological development and mental retardation.
The treatment of phenylketonuria is pretty straightforward: Limitation of
dietary phenylalanine. Tyrosine is plentifully available in a modern, proteinrich diet, so that the lack of endogenous formation won’t be a problem. The
challenge is then to diagnose the disease in newborn kids, before any damage
is done. Happily, the enzyme defect does not cause a problem during fetal
development, since both useful and potentially harmful metabolites are
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constantly equilibrated between the maternal and the fetal circulation.
Buildup of a metabolite in the fetus will therefore not occur as long as the
mother’s metabolism is able to degrade it.
CHAPTER 6: CARBOHYDRATE
Carbohydrates: Bountiful Sources of
Energy and Nutrients
What Are Carbohydrates?
♦One of the three macronutrients
♦Preferred energy source for the brain
♦Important source of energy for all cells
♦Composed of carbon, hydrogen, oxygen
♦Good sources: fruits, vegetables, and grains
Simple carbohydrates
� Contain one or two molecules
� Commonly referred to as sugars
Monosaccharides contain only one molecule
� Glucose, Fructose, Galactose
Disaccharides contain two molecules
� Lactose, Maltose, Sucrose
Complex carbohydrates
� Long chains of glucose molecules
� Starch, fiber, glycogen
Simple Carbohydrates – Monosaccharides
Glucose Fructose Galactose
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Simple Sugars – Dissacharides
Complex Carbohydrates
� Long chains of glucose molecules
� Hundreds to thousands of molecules long
� Also called polysaccharides
� Starch, glycogen, most Fibers
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Complex Carbohydrates
Starch
�Plants store carbohydrates as starch
�We digest (break down) starch to glucose
�Good sources: grains, legumes, and Tubers
Glycogen
� Animals store carbohydrates as glycogen
� Stored in the liver and muscles
� Not found in food and therefore not a source of dietary carbohydrate
Fiber
� Dietary fiber is the non-digestible part of plants
� Grains, seeds, legumes, fruits
� Functional fiber is carbohydrate extracted from plants or manufactured
� Total fiber = dietary + functional fiber
� Food labels only list dietary fiber
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Salivary amylase
� Enzyme that begins carbohydrate digestion in the mouth
� Breaks carbohydrates down to maltose
Carbohydrate digestion does not occur in the
stomach. Stomach acids inactivate salivary amylase Most chemical digestion of
carbohydrates occurs in the small intestine.
Pancreatic amylase
� Enzyme produced in the pancreas and secreted into the small intestine
Digests carbohydrates to maltose
Additional enzymes in the small intestine digest disaccharides to monosaccharides
� Maltase – breaks down maltose into two units of glucose
� Sucrase – breaks down sucrose into glucose & fructose
� Lactase – breaks down lactose into glucose & galactose
Monosaccharides are absorbed into the cells lining the small intestine and then enter the
bloodstream.
All monosaccharides are converted to glucose by the liver.
Glucose circulating in the blood is our primary energy source.
Excess glucose is converted to glycogen by the LIVER
We do not have the enzymes necessary to digest fiber.
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Bacteria in the large intestine can break down (ferment) some fiber. Most fiber remains
undigested and is excreted in the faeces
Glucose Utilization
Blood Glucose Regulation
Blood glucose level must be closely regulated.
Hormones control blood glucose levels:
� Insulin
� Glucagon
� Epinephrine
� Norepinephrine
� Cortisol
Growth hormone
Blood Glucose Regulation Insulin
Produced by beta cells of the pancreas
Stimulates glucose transporters (carrier proteins)
to help take glucose from the blood across the cell membrane
Stimulates the liver to take up glucose and convert to glycogen
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Blood Glucose Regulation Glucagon
Produced by alpha cells of the pancreas
Stimulates the liver to breakdown glycogen to glucose, making glucose available to body
cells Stimulates the breakdown of body proteins to amino acids to form new glucose Gluconeogenesis
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TUTORIAL 4, ENV416/400
A. Carbohydrates
1. Name the monosaccarides produced from the hydrolysis of the dissaccharides
below:
i)
Sucrose
ii)
Lactose
2. Explain the difference between glucose and fructose in terms of their structure.
3. D-Allosa, an aldohexose, has the same structure as D-glucose except that the
carboxyl at C3 is at the plane below in the cyclic hemiacetyl form. Draw the
structure of β-cyclic for D-allosa.
4. Name the two component of starch. State the similarity and difference between
these two structures.
5. Draw the open chain structure (Fischer projection) for D- fructose. Indicate the
carbonyl ketone or aldehyde group on the molecule.
6. Draw the cyclic β-D-fructose (Haworth structure).
B. Nitrogen Metabolism
1. Write the transamination reaction between α-ketoglutarate and alanine.
2. Write an equation for the net reaction of the urea cycle. Show how the urea cycle
is linked to the citric acid cycle.
3. Which aa in the urea cycle are the links to the citric acid cycle? Show how these
links occur.
4. How many ATP’s are required for one round of the urea cycle? Where do these
ATPs get used?
5. What species excrete excess nitrogen as ammonia? Which ones excrete as uric
acid?
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CHAPTER 7 :Metabolic processes central to ATP synthesisGlycolysis and Citric acid cycle
Glycolysis
Purpose: catabolism of glucose to provide ATPs and NADH molecules
Also provides building blocks for anabolic pathways.
Sequence of 10 enzyme-catalyzed reactions:
glucose
pyruvate
2 ATPs and 2 NADH produced
All enzymes (and reactions) are cytosolic.
Net reaction:
+
glucose + 2ADP + 2NAD +2Pi
2 pyruvate + 2ATP + 2NADH
+
+2H +2H2O
Can catabolize sugars other than glucose:
e.g. fructose ----> 2 glyceraldehyde 3-phosphate
e.g. lactose --> glucose + galactose
galactose --> glucose 1-phosphate --> glucose 6-phosphate
e.g. mannose ---> mannose 6-phosphate --> fructose 6-phosphate
Ten Steps of Glycolysis
1) glucose --> glucose 6-phosphate by hexokinase
G = -8.0 kcal/mole
Hexokinase also works on mannose and fructose at increased [ ].
Serves to trap glucose in the cell --> a phosphorylated molecule cannot leave
2) glucose 6-phosphate --> fructose 6-phosphate by glucose 6-phosphate
isomerase
Example of aldose--> ketose isomerization.
Enzyme is very stereospecific.
Reaction is near equilibrium in cell --> not a control point in glycolysis
3) fructose 6-phosphate --> fructose 1,6-bisphosphate by phosphofructokinase-1
(PFK-1)
Reaction has G = -5.3 kcal/mole and is metabolically irreversible.
Represents the first committed step in glycolysis.
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4) fructose 1,6-bisphosphate --> dihydroxyacetone phosphate + glyceraldehyde
3-phosphate by fructose 1,6 bisphosphate aldolase.
5) DHAP --> glyceraldehyde 3-phosphate by triose phosphate isomerase
Also catalyzes aldose--> ketose conversion.
Rate is diffusion controlled (substrate is converted to product as fast as
substrate is encountered).
6) glyceraldehyde 3-phosphate --> 1,3-bisphosphoglycerate by glyceraldehyde 3phosphate dehydrogenase
One molecule of NAD+ is reduced to NADH --> respiratory chain
7) 1,3 bisphosphoglycerate --> 3-phosphoglycerate
Phosphoryl group transfer to ADP to form ATP.
Because phosphate group comes from a substrate molecule, called substrate
level phosphorylation
First ATP-generating step of glycolysis.
8) 3-phosphoglycerate --> 2-phosphoglycerate by phosphoglycerate mutase
Mutases are enzymes that transfer phosphoryl groups from one part of a
substrate molecule to another.
9) 2-phosphoglycerate --> phosphoenolpyruvate (PEP) by enolase (forms double
bond)
10) PEP --> pyruvate
Second time for substrate level phosphorylation.
Reaction is metabolically irreversible.
FATE OF PYRUVATE
+
Under anaerobic conditions, cells must be able to regenerate NAD or
glycolysis will stop.
Usually regenerated by oxidative phosphorylation, but that requires O 2.
There are 2 anaerobic pathways that use NADH and regenerate NAD+.
1) alcoholic fermentation
Conversion of pyruvate to ethanol
H
+
CO2
pyruvate
pyruvate
decarboxylase
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+
NADH NAD
acetaldehyde
ethanol
alcohol
dehydrogenase
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+
glucose +2Pi + 2ADP + 2H ---> 2 ethanol + 2CO2 + 2ATP + 2H2O
2) lactate fermentation
+
pyruvate
+
NADH + H
NAD
------------------------> lactate
lactate
dehydrogenase
glucose +2Pi + 2ADP ---> 2 lactate + 2ATP + 2H20
Lactate causes muscles to ache.
Also produced by bacterial fermentation of lactose.
3) entry into citric acid cycle
The Citric Acid Cycle
Summary:
Yields reduced coenzymes (NADH and QH2) and some ATP (2).
Preparative step is oxidative decarboxylation involving coenzyme A.
Occurs in eucaryotic mitochondrion and procaryotic cytosol.
How does the pyruvate get into the mitochondrion from the cytosol?
Pyruvate passes through channel proteins called porins (can transport
molecules < 10,000 daltons) located in outer mitochondrial membrane.
To get from intermembrane space to matrix involves pyruvate translocase
(symporter that also moves H+ into matrix).
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CONVERSION OF PYRUVATE TO ACETYL COA
Enzyme is pyruvate dehydrogenase complex, composed of three enzymes:
1) pyruvate dehydrogenase
2) dihydrolipoamide acetyltransferase
3) dihydrolipoamide dehydrogenase
Reaction occurs in 5 steps:
1) E1 uses TPP as a prosthetic group and decarboxylates pyruvate --> forms
HETPP intermediate
2) E1 then transfers acetyl group to oxidized lipoamide --> acetyllipoamide
3) E2 transfers acetyl group to coenzyme A to form acetyl CoA;
dihydrolipoamide becomes reduced
4) E3 reoxidizes lipoamide portion of E2; prosthetic group of E3 (FAD)
oxidizes reduced lipoamide --> FADH2
5) NAD+ is reduced by E3-FADH --> E3-FAD + NADH + H+
E2 acts like a crane by swinging substrate between protein complexes in enzyme.
Regulation of PDH complex:
Regulated by covalent modification by phosphorylation.
inactive = phosphorylated; active = dephosphorylated
E1
inhibited at high [ATP]; inhibited at high [GTP]
activated by high [AMP], high [Ca2+], high [pyruvate]
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E2
inhibited by high [acetyl CoA]
activated by high [CoA-SH]
E3
inhibited by high [NADH]
activated by high [NAD+]
THE CITRIC ACID CYCLE
Summary:
Composed of 8 reactions
4 carbon intermediates are regenerated
2 molecules of CO2 released (6C--> 4C)
Most of energy stored as NADH and QH2
1) citrate synthase
Irreversible reaction
Acetyl CoA reacts with oxaloacetate --> citrate and CoA
2) aconitase
Citrate --> isocitrate
3) isocitrate dehydrogenase
Irreversible reaction
Substrate first oxidized (2e- and H+ given to NAD+), then decarboxylated
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Isocitrate --> -ketoglutarate + CO2 + NADH + H+
4) -ketoglutarate dehydrogenase complex
-ketoglutarate first decarboxylated, oxidized (2e- and H+ given to NAD+),
and HS-CoA added
Product is succinyl CoA
Enzyme complex similar the PDH, but has dihydrolipoamide
succinyltransferase instead of acetyltransferase.
5) succinyl CoA synthetase or succinate thiokinase
succinyl CoA --> succinate
Substrate has high energy thioester bond; that energy is stored as nucleoside
triphosphate via substrate level phosphorylation
GDP +Pi --> GTP
mammals
ADP +Pi --> ATP
plants and bacteria
6) succinate dehydrogenase complex
Enzyme is embedded in inner mitochondrial membrane.
Has FAD covalently bound to it (prosthetic group).
Converts succinate --> fumarate with generation of FADH2 --> ETS
FAD is regenerated by reduction of a mobile molecule called ubiquinone
(coenzyme Q) --> QH2.
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CHAPTER 8 :Electron Transport and Oxidative Phosphorylation

Oxidative phosphorylation - process in which NADH and QH2 are oxidized
and ATP is produced.
Enzymes are found in inner mitochondrial membrane in eukaryotes.
In prokaryotes, enzymes are found in cell membrane.
Process consists of 2 separate, but coupled processes:
1) respiratory electron-transport chain
 Responsible for NADH and QH2 oxidation
 Final e- acceptor is molecular oxygen
 Energy generated from electron transfer is used to
pump H+ into intermembrane space from matrix --->
matrix becomes more alkaline and negatively charged.
2) ATP synthesis
 Proton concentration gradients represents stored
energy
+
 When H are moved back across inner mitochondrial
membrane through ATP synthase ---> ADP is
phosphorylated to form ATP



Chemiosmotic Theory of ATP Production
Proposed by Peter Mitchell in 1961 (won Nobel Prize for this work).
Proton concentration gradient serves as energy reservoir for ATP synthesis.
Proton concentration gradient also known as proton motive force (PMF).
Components of Electron Transport System
There are 5 protein complexes:
I) NADH-ubiquinone oxidoreductase
II) succinate-ubiquinone oxidoreductase
III) ubiquinol-cytochrome c oxidoreductase
IV) cytochrome c oxidase
V) ATP synthase


Electrons flow through ETS in direction of increasing reduction potential.
Two mobile electron carriers also involved: ubiquinone (Q) between
complexes I or II and III, and cytochrome c between complexes III and IV.
Electrons enter ETS 2 at a time from either NADH or succinate.

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I - NADH-ubiquinone oxidoreductase
Transfers 2e from NADH to Q as hydride ion (H-)
First electron transferred to FMN --> FMNH2 ---> Fe-S cluster ---> Q
+
-
Also pumps 4H /2e into intermembrane space
II - succinate-ubiquinone oxidoreductase
Transfers e from succinate to Q
First transferred to FAD ---> FADH2 ---> 3 Fe-S clusters ---> Q
Not enough energy to contribute to proton gradient via proton pumping
III - ubiquinol-cytochrome c oxidoreductase
Rransfers e from QH2 to cytochrome c facing intermembrane space
Composed of 9-10 subunits including 2 Fe-S clusters, cytochrome b560,
cytochrome b566, and cytochrome c1.
+
Transports 2H from matrix into intermembrane space
IV - cytochrome c oxidase
Contains cytochromes a and a3
Contributes to proton gradient in two ways:
+
1) pumps 2H for each pair of e transferred (per O2 reduced)
+
+
2) consumes 2H when oxygen is reduced to H2O ---> lowers [H ]matrix
Carbon monoxide (CO) and cyanide (HCN) bind here
V - ATP synthase
+
Does not contribute to H gradient, but helps relieve it
Also called FOF1 ATP synthase
F1 component contains catalytic subunits
FO component is proton channel that is transmembrane
+
Per ATP synthesized, 3H move through ATP synthase
oligomycin - antibiotic that binds to channel and prevents proton entry --> no
ATP synthesized
TRANSPORT OF MOLECULES ACROSS MITOCHONDRIAL MEMBRANE


Inner mitochondrial membrane is impermeable to NADH and NAD +.
Must use a shuttle to regenerate NAD+ for glycolysis; solution is to shuttle
electrons across membrane, rather than NADH itself.
There are two shuttles in operation:
1) glycerol phosphate shuttle
 Found in insect flight muscles and mammalian cells in which
high rates of oxidative phosphorylation must occur

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
Cytosolic glycerol 3-phosphate dehydrogenase converts
DHAP to glycerol 3-phosphate
 Converted back to DHAP by membrane-bound glycerol 3phosphate dehydrogenase
 Result is transfer to 2e to FAD --> Q ---> complex III
 Produces fewer ATP molecules (1.5 vs. 2) because complex
I is bypassed
2) malate-aspartate shuttle
 Found in liver and heart
 Cytosolic NADH reduces oxaloacetate --> malate -->
transported via dicarboxylate translocase into matrix
 In matrix, malate --> oxaloacetate --> aspartate --->
transported out via glutamate-aspartate translocase
 Converted back to oxaloacetate.......
 No reduction in ATP yield
Must also be able to transport other metabolites into and out of matrix:
1) ADP/ATP carrier or ADP/ATP translocase
Adenine nucleotide translocase which exchanges ADP and ATP
(antiporter)
+
2) Pi/H carrier
+
Couples inward movement of Pi with symport of H from gradient
REGULATION OF OXIDATIVE PHOSPHORYLATION



Depends upon substrate availability and energy demands in the cell.
Important substrates are NADH, O2, and ADP.
As ATP is used, more ADP is available, translocated through adenine
nucleotide translocase --> electron transport increases.
Known as respiratory control.
Helps to replenish ATP pool in the cell, which is kept nearly constant.
Rates of glycolysis, citric acid cycle, and electron transport system are
matched to a cell’s ATP requirements.
Proton gradient can be short-circuited to generate heat
Found in brown adipose tissue in newborn mammals and animals that
hibernate, and animals adapted to cold conditions
A protein called thermogenin forms a proton channel in inner
mitochondrial membrane --> dissipates proton gradient, but electrons still
flow --> heat production
Pathway is activated by fatty acids from triacylglycerol catabolism from
epinephrine stimulation







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Superoxide Production
Even though cytochrome oxidase and other proteins that reduce oxygen have
been designed not to release O2.- (superoxide anion), it still does happen.
Protonation of superoxide anion yields hydroperoxyl radical (HO 2.), which can
react with another molecule to produce H2O2.
Enzyme superoxide dismutase catalyzes this reaction
+
2H
O2.- + O2.- ----------------------------> H2O2 + O2
superoxide dismutase
Recent findings have indicated that superoxide dismutase mutations can
cause amyotrophic lateral sclerosis (Lou Gehrig’s disease), in which motor
neurons in brain and spinal cord degenerate.
The hydrogen peroxide formed is scavenged by catalase:
H2O2 + H2O2
2H2O + O2
catalase
Peroxidases catalyze an analogous reaction:
ROOH + AH2
ROH + H2O + A
peroxidase
7) fumarase
fumarate --> malate
8) malate dehydrogenase
L-malate --> oxaloacetate
2e- and H+ given to NAD+ --> NADH
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Net reaction for citric acid cycle:
acetyl CoA + 3NAD+ + Q + GDP(ADP)+ Pi +2H2O ---> HS-CoA + 3NADH + QH2 +
GTP(ATP) + 2CO2 +
2H+
Energy Budget so far from 1 molecule of glucose:
glycolysis
2 ATP
Prep Step
TCA
2 NADH
2 NADH
2 ATP
4 ATP
6 NADH
2 QH2
10 NADH
ATP Production:
glycolysis
2 ATP
Prep Step
TCA
6 ATP equivalents
6 ATP equivalents
2 ATP
18 ATP equivalents + 4 ATP equivalents
4 ATP
34 ATP
substrate (ox. phos.)
level phos.
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REGULATION OF TCA CYCLE
There are 2 enzymes that are regulated:
1) isocitrate dehydrogenase
allosterically activated by high [Ca2+] and high [ADP]
allosterically inhibited by high [NADH]
2) -ketoglutarate dehydrogenase
allosterically activated by high [Ca2+]
allosterically inhibited by high [NADH] and high [succinyl CoA]
ENTRY AND EXIT OF METABOLITES
Citrate, -ketoglutarate, succinyl CoA, oxaloacetate lead to biosynthetic pathways.
Citrate --> fatty acids and sterols in liver and adipocytes
(cleaved into acetyl CoA if needed)
-ketoglutarate --> glutamate --> amino acid synthesis or nucleotide synthesis
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succinyl CoA --> propionyl CoA --> fatty acid synthesis
--> porphyrin synthesis
oxaloacetate --> gluconeogenesis
--> asparate --> urea synthesis, a.a. synthesis, pyrimidine synthesis
Pathway intermediates must be replenished by anapleurotic reactions.
GLYOXYLATE CYCLE
Modification of citric acid cycle.
Anabolic pathway in plants, bacteria, yeast.
Takes 2 carbon compounds and converts them to glucose.
Common in plants which store energy reserves as oils, but must be converted to
carbohydrates during germination.
In eucaryotes, a glyoxysome is a special organelle where this occurs.
Gluconeogenesis, the Pentose Phosphate Pathway and Glycogen
Metabolism
GLYCOGEN METABOLISM

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Glycogen stored in muscle and liver cells.
Important in maintaining blood glucose levels.
Glycogen structure:  1,4 glycosidic linkages with  1,6 branches.
Branches give multiple free ends for quicker breakdown or for more places to add
additional units.
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Glycogen Degradation

Glucose residues of starch and glycogen released through enzymes called starch
phosphorylases and glycogen phosphorylases.
Catalyze phosphorolosis:

polysaccharide +Pi ---> polysaccharide(n-1) + glucose 1-phosphate

Pyridoxal phosphate (PLP) is prosthetic group in active site of enzyme; serves as a
proton donor in active site.
Allosterically inhibited by high [ATP] and high [glucose 6-phosphate].
Allosterically activated by high [AMP].
Sequentially removes glucose residues from nonreducing ends of glycogen, but
stops 4 glucose residues from branch point --> leaves a limit dextran.
Limit dextran further degraded by glycogen-debranching enzyme
(glucanotransferase activity) which relocated the chain to a free hydroxyl end.
Amylo-1,6-glucosidase activity of debranching enzyme removes remaining
residues of
chain.
This leaves substrate for glycogen phosphorylase.
Each glucose molecule released from glycogen by debranching enzyme will yield
3 ATPs in glycolysis.
Each glucose molecule released by glycogen phosphorylase will yield 2 ATPs in
glycolysis.
Why?
 ATP not needed in first step because glucose 1-phosphate already
formed.
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phosphoglucomutase
glucose 1-phosphate ----------------------> glucose 6-phosphate
1) In liver, kidney, pancreas, small intestine,
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glucose 6-phosphatase
glucose 6-phosphate --------------------------> glucose + Pi
Glycogen Synthesis



Not reverse of glycogen degradation because different enzymes are used.
About 2/3 of glucose ingested during a meal is converted to glycogen.
First step is the first step of glycolysis:
hexokinase
glucose --------------> glucose 6-phosphate

There are three enzyme-catalyzed reactions:
phosphoglucomutase
glucose 6-phosphate ---------------------> glucose 1-phosphate
glucose 1-phosphate ---------------> UDP-glucose (activated form of
glucose)
glycogen synthase
UDP-glucose ----------------------> glycogen

Glycogen synthase cannot initiate glycogen synthesis; requires preexisting primer
of glycogen consisting of 4-8 glucose residues with  (1,4) linkage.
Protein called glycogenin serves as anchor; also adds 7-8 glucose residues.
Addition of branches by branching enzyme (amylo-(1,4 --> 1,6)transglycosylase).
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Takes terminal 6 glucose residues from nonreducing end and attaches it via
(1,6) linkage at least 4 glucose units away from nearest branch.
REGULATION OF GLYCOGEN METABOLISM
Mobilization and synthesis of glycogen under hormonal control.
Three hormones involved:
1) insulin


51 a.a. protein made by  cells of pancreas.
Secreted when [glucose] high --> increases rate of glucose transport into muscle
and fat via GLUT4 glucose transporters.
Stimulates glycogen synthesis in liver.

2) glucagon

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
29 a.a. protein secreted by  cells of pancreas.
Operational under low [glucose].
Restores blood sugar levels by stimulating glycogen degradation.
3) epinephrine
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
Stimulates glycogen mobilization to glucose 1-phosphate --> glucose 6-phosphate.
Increases rate of glycolysis in muscle and the amount of glucose in bloodstream.
Occurs in response to fight-or-flight response.
Binds to -adrenergic receptors in liver and muscle and 1 receptors in liver cells.
Binding of epinephrine or glucagon to  receptors activates adenylate cyclase,
which is a membrane-traversing enzyme that converts ATP --> cAMP -->
activates protein kinase A.
Binding of epinephrine to 1 receptors activates IP3 pathway --> protein kinase C
--> phosphorylation of insulin receptors -> insulin cannot bind.
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Regulation of glycogen phosphorylase and glycogen synthase

Reciprocal regulation.
 Glycogen synthase -P --> inactive form (b).
 Glycogen phosphorylase-P ---> active (a).
When blood glucose is low, protein kinase A activated through hormonal action
of glucagon --> glycogen synthase inactivated and phosphorylase kinase
activated --> activates glycogen phosphorylase --> glycogen degradation
occurs.
Phosphorylase kinase also activated by increased [Ca2+] during muscle contraction.
To reverse the same pathway involves protein phosphatases, which remove
phosphate groups from proteins --> dephosphorylates phosphorylase
kinase and glycogen phosphorylase (both inactivated), but
dephosphorylation of glycogen synthase activates this enzyme.
Protein phosphatase-1 activated by insulin --> dephosphorylates glycogen
synthase --> glycogen synthesis occurs.
In liver, glycogen phosphorylase a inhibits phosphatase-1 --> no glycogen
synthesis can occur.
Glucose binding to protein phosphatase-1 activated protein phosphatase-1 -->
it dephosphorylates glycogen phosphorylase --> inactivated --> no
glycogen degradation.
Protein phosphatase-1 can also dephosphorylate glycogen synthase --> active.
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GLUCONEOGENESIS
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Synthesis of glucose from noncarbohydrate sources.
Major precursors are lactate and alanine in the liver and kidney.
lactate - active skeletal muscles
glycerol - lipid catabolism
amino acids - diet and protein catabolism
Used to maintain blood glucose levels when glycogen supplies are low or depleted.
Major site of occurrence is the liver, but also occurs in kidney.
Designed to make sure blood glucose levels are high enough to meet the demands
of brain and muscle (cannot do gluconeogenesis).
NOT the reverse of glycolysis. Why?
PFK, PK, and hexokinase catalyze metabolically irreversible steps.
Solution: by-pass these steps, but use all the other enzymes.
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1) pyruvate ---> phosphoenolpyruvate
ATP ADP + Pi
GTP
GDP
pyruvate ------------------> oxaloacetate -----------------> PEP
HCO3- pyruvate
PEP carboxykinase
carboxylase
TCA
Cycle
Pi
2) fructose 1,6 bisphosphate
fructose 6-phosphate
fructose 1,5-bisphosphatase
glucose 6-phosphatase
3) glucose 6-phosphate --------------------------> glucose
This enzyme is bound to ER membrane, but faces ER lumen.
GLUT7 transporter must transport glucose 6-phosphate into ER lumen.
Enzyme not found in membrane of brain or muscle
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PRECURSORS FOR GLUCONEOGENESIS
1) lactate
Cori cycle - no net gain or loss of glucose
Anaerobic respiration of pyruvate.
2) amino acids
glutamate -ketoglutarate
pyruvate -----------------------------------> alanine
transamination
3) glycerol
glycerol kinase
glycerol ------------------> glycerol 3-phosphate -----> DHAP
If glycerol 3-phosphate dehydrogenase is embedded in inner mitochondrial
membrane, e- passed to ubiquinone.
If enzyme is cytosolic, NADH is also a product.
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REGULATION OF GLUCONEOGENSIS
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
Glycolysis and gluconeogenesis are reciprocally regulated.
If both pathways were activated, e.g.
fructose 6-phosphate + ATP ------> fructose 1,6-bisphosphate + ADP
fructose 1,6-bisphosphate + H2O ---> fructose 6-phosphate + Pi
net reaction: ATP + H2O ---> ADP + Pi


Called substrate cycle ---> “burn” 4 ATPs for every 2 ATPs made (can be used to
generate heat).
Reason why enzymes are regulated --> prevents this from happening.

Two regulatory points are the two steps which had different enzymes.
fructose 1,6-bisphosphatase
inhibited by AMP and fructose 2,6-bisphosphate
pyruvate carboxylase
activated by acetyl CoA
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PENTOSE PHOSPHATE PATHWAY
Provides NADPH (serves as e- donor) and forms ribose 5-phosphate (nucleotide
synthesis).
Pathway active is tissues that synthesize fatty acids or sterols because large amounts of
NADPH needed.
In muscle and brain, little PPP activity.
All reactions are cytosolic.
Divided into 2 stages:
1) oxidative
glucose 6-phosphate +2 NADP+ + H2O --> ribulose 5-phosphate +
2 NADPH + CO2 + 2H+
2) nonoxidative
Uses transketolases (transfers 2-C units) and transaldolases
(transfers 3-C units).
Links PPP with glycolysis.
Used to catalyze these types of reactions:
C5 + C5 <----> C7 + C3
C7 + C3 <----> C4 + C6
C5 + C4 <----> C3 + C6
All reactions are reversible --> very flexible pathway.
Example:

If ribose 5-phosphate needed, fructose 6-phosphate + glyceraldehyde 3-phosphate
taken from glycolysis and channeled through PPP to make product.
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If NADPH is needed, then ribulose 5-phosphate is converted to glyceraldehyde 3phosphate and fructose 6-phosphate --> converted to glucose 6-phosphate -->
more NADPH made.
If use PPP, 1 glucose can be completely oxidized to 12 NADPH and 6 CO2.
If NADPH and ATP are needed, ribulose 5-phosphate converted into
glyceraldehyde 3-phosphate and fructose 6-phosphate --> glycolysis -->
pyruvate.
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REGULATION OF PENTOSE PHOSPHATE PATHWAY


Controlled by levels of NADP+.
Controlled step is dehydrogenation of glucose 6-phosphate to 6phosphogluconolactone.
Enzyme stimulated by high [NADP+].
Nonoxidative branch controlled primarily by substrate availability.
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Health Disorders
Three health disorders related to carbohydrate
metabolism are
� Diabetes
� Hypoglycemia� Lactose intolerance
Diabetes
Inability to regulate blood glucose levels
Three types:
� Type 1 diabetes
� Type 2 diabetes
� Gestational diabetes
Uncontrolled diabetes can cause nerve damage,
kidney damage, blindness, and can be fatal
Diabetes – Type 1
Accounts for 10% of all cases
Patients do not produce enough insulin
Causes hyperglycemia (high blood glucose)
Requires insulin injections
May be an autoimmune disease
Once known as juvenile-onset diabetes or insulin dependent diabetes mellitus (IDDM)
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Diabetes – Type 2
Most diabetics have type 2 diabetes
Progressive disease with biological changes occurring over time
Body cells become resistant, or less responsive to insulin Hyperglycemia results when
cells cannot take in the glucose from the blood
Once known as non-insulin dependent diabetes mellitus (NIDDM)
Diabetes – Type 2
Cause is unclear but genetics, obesity, and physical inactivity play a large role
Treat with weight-loss diet, regular exercise, and, if necessary, medications
Healthy lifestyle choices may prevent or delay the onset of type 2 diabetes:
� Balanced diet and regular exercise
� Achieving and maintaining healthy body weight
Hypoglycemia
Low blood sugar (glucose)
Causes shakiness, sweating, anxiety
Reactive hypoglycemia: pancreas secretes too much insulin after a high-carbohydrate
meal Fasting hypoglycemia: pancreas produces too much insulin, even when someone has
not eaten.
Lactose Intolerance
Insufficient enzyme lactase to digest the lactose containing
foods
Symptoms: gas, bloating, cramping, diarrhea
Extent of intolerance: mild to severe
Persons with lactose intolerance may need to find alternate sources of calcium
CHAPTER 9 :Lipids and Membranes
Lipids are water-insoluble that are either hydrophobic (nonpolar) or amphipathic
(polar and nonpolar regions).
There are many types of lipids:
1) fatty acids

The simplest with structural formula of R-COOH where R = hydrocarbon
chain.
They differ from each other by the length of the tail, degree of unsaturation,
and position of double bonds.
pKa of -COOH is 4.5-5.0 --> ionized at physiological pH.
If there is no double bond, the fatty acid is saturated.
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If there is at least one double bond, the fatty acid is unsaturated.
Monounsaturated fatty acids contain 1 double bond; polyunsaturated fatty
acids have >2 double bonds.
n represents where double bond occurs as you
count from the carboxyl end (see Table 9.1).

e.g.

-enoate
one double bond
-dienoate
2
“
-trienoate
3
“
-tetraenoate
4
“
Can also use a colon separating 2 numbers, where the first number
represents the number of carbon atoms and the second number indicates
the location of the double bonds.
e.g. linoleate

9,12
or cis,cis -
9,12octadecadienoate
Physical properties differ between saturated and unsaturated fatty acids.
Saturated = solid at RT; often animal source; e.g. lard
Unsaturated = liquid at RT; plant source; e.g. vegetable oil


The length of the hydrocarbon tails influences the melting point.
As the length of tails increases, melting points increases due to number of
van der Waals interactions.
Also affecting the melting point is the degree of unsaturation.
As the degree of unsaturation increases, fatty acids become more fluid-->
melting point decreases ( kinks in tails decrease number of van der Waals
interactions).
Fatty acids are also an important sources of energy.



9 kcal/g vs. 4 kcal/g for carbohydrates and proteins.
2) triacylglycerols


Also called triglycerides.
Made of 3 fatty acyl residues esterified to glycerol.
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Very hydrophobic, neutral in charge ---> can be stored in anhydrous form.
Long chain, saturated triacylglycerols are solid at RT (fats).
Shorter chain, unsaturated triacylglycerols are liquid at RT (oils).
Lipids in our diet are usually ingested as triacylglycerols and broken down
by lipases to release fatty acids from their glycerol backbones
Also occurs in the presence of detergents called bile salts.
 Form micelles around fatty acids that allow them to be
absorbed by
intestinal epithelial cells.
 Transported through the body as lipoproteins.

3) glycerophospholipids




Main components of cell membranes.
Are amphipathic and form bilayers.
All use glycerol 3-phosphate as backbone.
Simplest is phosphatidate = 2 fatty acyl groups esterified to glycerol 3phosphate.
Often, phosphate is esterified to another alcohol to form...
 phosphatidylethanolamine
 phosphatidylserine
 phosphatidylcholine


Enzymes called phospholipases break down biological membranes.
 A-1 = hydrolysis of ester bond at C-1.
 A-2 = hydrolysis of ester bond at C-2; found in pancreatic
juice.
 C = hydrolysis of P-O bond between glycerol and phosphate
to create phosphatidate.
 D = same
4) sphingolipids



Second most important membrane constituent.
Very abundant in mammalian CNS.
Backbone is sphingosine (unbranched 18 carbon alcohol with 1 trans
C=C
between C-4 and C-5), NH3+ group at C-2, hydroxyl
groups at C-1 and C-3.
Ceramides are intermediates of sphingolipid synthesis.
There are three families of sphingolipids:
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1) sphingomyelin - phosphocholine attached to C-1 hydroxyl
group of ceramide; present in the myelin sheaths around some
peripheral nerves.
2) 2)cerebrosides - glycosphingolipid; has 1 monosaccharide
-glycosidic linkage to C-1 of ceramide;
most common is galactocerebroside, which is abundant in
nervous tissue.
3) gangliosides - glycosphingolipid containing N-acetylneuraminic
acid; present on all cell surfaces.
Hydrocarbon tails embedded in membrane with oligosaccharides
facing extracellularly.
Probably used as cell surface markers, e.g. ABO blood group
antigens.
Inherited defects in ganglioside metabolism --> diseases, such as TaySachs disease.
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5) steroids


Called isoprenoids because their structure is similar to isoprene.
Have 4 fused rings: 3 6-membered rings (A,B,C) and 1 5-membered ring
(D).
Cholesterol is an important component of cell membranes of animals, but
rare in plants and absent in procaryotes.
Also have mammalian steroid hormones (estrogen, androgens) and bile
salts.
Differ in length of side chain at C-17, number and location of methyl groups,
double bonds, etc.
Cholesterol’s role in membranes is to broaden the phase transition of
cell membranes ---> increases membrane fluidity because
cholesterol disrupts packing of fatty acyl chains.




6) other lipids not found in membranes

waxes - nonpolar esters of long chain fatty acids and alcohols
very water insoluble
high melting point --> solid at outdoor/RT.
Roles: protective coatings of leaves, fruits, fur, feathers,
exoskeletons.
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eicosanoids - 20 carbon polyunsaturated fatty acids
e.g. prostaglandins - affect smooth muscle --> cause
constriction; bronchial constriction of asthmatics; uterine
contraction during labo
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
limonene - smell of lemons
bactoprenol - involved in cell wall synthesis
juvenile hormone I - larval development of insects
Biological Membranes




Central transport of ions and molecules into and out of the cell.
Generate proton gradients for ATP production by oxidative phosphorylation.
Receptors bind extracellular signals and transduce the signal to cell interior.
Structure:
 Glycerophospholipids and glycosphingolipids form bilayers.
 Noncovalent interactions hold lipids together.
 5-6 nm thick and made of 2 leaflets to form a lipid bilayer driven by
hydrophobic effects.
 About 40% lipid and 50% proteins by mass, with about 10%
carbohydrates.
Protein and lipid composition varies among membranes but all have same
basic structure --> Singer and Nicholson fluid mosaic model in 1972.

Membrane fluidity:



Can undergo transverse diffusion (one leaflet to another) but very rare.
Membrane has an asymmetrical lipid distribution that is maintained by
flippases or translocases that are ATP-driven.
In 1970, Frye and Edidin demonstrated that proteins are also capable of
diffusion by using heterocaryons, but occurs at a rate that is 100-500 times
slower than lipids.
Most membrane protein diffusion is limited by aggregation or attachment to
cytoskeleton.
Can examine distribution of membrane proteins by freeze-fracture electron
microscopy.
Membrane fluidity is dependent upon the flexibility of fatty acyl chains.
 Fully extended saturated fatty acyl chains show maximum van der
Waals interactions.
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 When heated, the chains become disordered --> less interactions -> membrane “shrinks” in size due to less extension of tails --> due to
rotation around C-C bond.
 For lipids with unsaturated acyl chains, kink disrupts ordered
packing and increases membrane fluidity --> decreases phase transition
temperature (becomes more fluid at lower temperature).
 Some organisms can alter their membrane fluidity by adjusting the
ratio of
unsaturated to saturated fatty acids.
e.g. bacteria grown at low temperature increase the
proportion of unsaturated fatty acyl groups.
e.g. warm-blooded animals have less variability in that ratio
because of the lack of temperature fluctuations.
exception: reindeer leg has increased number of fatty acyl
groups as get closer to hoof --> membrane can remain more fluid at lower
temperatures.

Cholesterol also affects membrane fluidity.
 Accounts for 20-25% of lipid mass of membrane.
 Broadens the phase-transition temperature.
 Intercalation of cholesterol between membrane lipids restricts
mobility of fatty acyl chains ---> fluidity decreases.
 Helps maintain constant membrane fluidity despite changes
in temperature and degree of fatty acid saturation.
CHAPTER 10 : Lipid Metabolism
Fatty acids have four major physiologic roles in the cell:
 Building blocks of phospholipids and glycolipids
 Added onto proteins to create lipoproteins, which targets them to
membrane locations
 Fuel molecules - source of ATP
 Fatty acid derivatives serve as hormones and intracellular messengers
Absorption and Mobilization of Fatty Acids
 Most lipids are triacylglycerols, some are phospholipids and cholesterol.
 Digestion occurs primarily in the small intestine.
 Fat particles are coated with bile salts (amphipathic) from gall bladder.
 Degraded by pancreatic lipase (hydrolyzes C-1 and C-3 ---> 2 fatty acids
and 2-monoacylglycerol).
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Can then be absorbed by intestinal epithelial cells; bile salts are
recirculated after being absorbed by the intestinal epithelial cells.
In the cells, fatty acids are converted by fatty acyl CoA molecules.
Phospholipids are hydrolyzed by pancreatic phospholipases, primarily
phospholipase A2.
Cholesterol esters are hydrolyzed by esterases to form free cholesterol,
which is solubilized by bile salts and absorbed by the cells.
Lipids are transported throughout the body as lipoproteins.
Lipoproteins consist of a lipid (tryacylglycerol, cholesterol, cholesterol
ester) core with amphipathic molecules forming layer on outside.
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

Lipoproteins

Both transported in form of lipoprotein particles, which solubilize
hydrophobic lipids and contain cell-targeting signals.
 Lipoproteins classified according to their densities:
o chylomicrons - contain dietary triacylglycerols
o chylomicron remnants - contain dietary cholesterol esters
o very low density lipoproteins (VLDLs) - transport endogenous
triacylglycerols, which
are hydrolyzed by lipoprotein lipase at
capillary surface
o intermediate-density lipoproteins (IDL) - contain endogenous
cholesterol esters, which are taken up by liver cells via receptormediated endocytosis and converted to LDLs
o low-density lipoproteins (LDL) - contain endogenous cholesterol
esters, which are taken up by liver cells via receptor-mediated
endocytosis; major carrier of cholesterol in blood; regulates de novo
cholesterol synthesis at level of target cell
o high-density lipoproteins - contain endogenous cholesterol esters
released from dying cells and membranes undergoing turnover
Storage of Fatty Acids
 Triacylglycerols are transported as chylomicrons and VLDLs to adipose
tissue; there, they are hydrolyzed to fatty acids, which enter adipocytes
and are esterified for storage.
 Mobilization is controlled by hormones, particularly epinephrine, which
-adrenergic receptors on adipocyte membrane --> protein kinase
A activated --> phosphorylates hormone-sensitive lipase --> converts
triacylglycerols to free fatty acids and monoacylglycerols.
 Insulin inhibits lipid mobilization (example of reciprocal regulation).
 Monoacylglycerols formed are phosphorylated and oxidized to DHAP
(intermediate of glycolysis and gluconeogenesis).
ATP
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glycerol
glycerol 3-phosphate
phosphate
glycerol kinase
2009
dihydroxyacetone
glycerol phosphate
dehydrogenase
Can be converted to glucose (gluconeogenesis) or pyruvate (glycolysis) in
the liver.
Fa
-oxidation)

-oxidation.
 Pathway that removes 2-C units at a time --> acetyl CoA --> citric acid
cycle --> ATP

-oxidation:
o Activation of fatty acids in cytosol catalyzed by acyl CoA synthetase;
two high energy bonds are broken to produce AMP
o 2) Transport of fatty acyl CoA into mitochondria via carnitine shuttle
o
-oxidation - cyclic pathway in which many of the same enzymes
are used repeatedly (see pathway sheet)
-oxidation of odd chain and unsaturated fatty acids

-oxidation until propionyl CoA is formed.
 Propionyl CoA is then converted to succinyl CoA, which then enters the
Krebs cycle.
 See pathway sheet for details

-

oxidation.
o enoyl-CoA isomerase
o 2,4-dienoyl-CoA reductase
How the pathway looks depends upon the location of the double bond, but
there are two possibilities.
See pathway sheets for details.

ATP generation from Fatty Acid Oxidation:

Can be estimated from the amount of acetyl CoA, QH2, and NADH
produced.
See pathway sheet.

Regulation of Fatty Acid Oxidation

Already talked about fatty acid mobilization via epinephrine.
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-oxidation.
Both molecules allosterically inhibit pyruvate dehydrogenase complex.
Most of acetyl CoA produced goes to Krebs cycle; during periods of
fasting, excess acetyl CoA is produced, too much for Krebs cycle.
Also in diabetes, oxaloacetate is used to form glucose by gluconeogenesis
--> concentration of oxaloacetate is lowered.
Result is the diversion of acetyl CoA to form acetoacetate and 3hydroxybutyrate; these two molecules plus acetone are known as ketone
bodies.
Acetoacetate is formed via the following reactions:



acetyl
CoA
2 acetyl CoA
HMG-CoA lyase
CoA
3-hydroxy-
acetyl CoA
acetoacetate
3-methylglutaryl CoA
NADH + H+
NAD+
-hydroxy
H+
butyrate
CO2
Dehydrogenase
3-hydroxybutyrate
acetone

The major site of ketone body synthesis is the liver, within the
mitochondrial matrix ---> transported to the bloodstream.
Acetoacetate and 3-hydroxybutyrate are used in respiration and are
important sources of energy.
Cardiac muscle and the renal cortex perferentially use acetoacetate over
glucose.
Glucose is used by brain and RBCs; in brain, ketone bodies substitute for
glucose as fuel because the brain cannot undergo gluconeogenesis.
Acetoacetate can be converted to acetyl CoA and oxidized in citric acid
cycle only in nonhepatic tissues.
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
Diabetes (insulin-dependent diabetes mellitus; IDDM)
Decreased insulin secretion by beta cells of pancreas; could be caused by
viruses (?)
Juvenile onset
Patients are thin, hyperglycemic, dehydrated, polyuric (pee a lot), hungry,
thirsty
In these patients, glycogen mobilization, gluconeogenesis, fatty acid oxidation
occurs --- > massive ketone body production; also, some of the glucose is in
urine (tends to pull water out of body) ----> diabetic ketoacidosis
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FATTY ACID SYNTHESIS
Important features of this pathway:
-oxidation takes place in mitochondrial
matrix.
2) Intermediates are bound to sulfhydral groups of acyl carrier protein (ACP);
-oxidation are bonded to CoA
3) Growing fatty acid chain is elongated by sequential addition of two-carbon
units derived from acetyl CoA
+
-oxidation are NAD
and FAD
5) Elongation of fatty acid stops when palmitate (C16) is formed; further
elongation and insertion of double bonds carried out later by other enzymes
Fatty acid synthesis takes place in three stages:
1) Mitochondrial acetyl CoA is transported into cytosol via citrate transport
system Acetyl CoA is condensed with oxaloacetate to form citrate --->
antiported out with inward movement of anion
Citrate cleaved by cytosolic citrate lyase --> oxaloacetate + acetyl CoA
2) Formation of malonyl CoA
Acetyl CoA carboxylase is key regulatory enzyme
Influenced by glucagon --> inactivates enzyme in liver
Epinephrine inactivates enzyme in adipocytes
Citrate allosterically activates enzyme
Fatty acyl CoA allosterically inhibits enzyme
3) Assembly of fatty acid chain via fatty acid synthase
Consists of five separate stages:
1) Loading - acetyl CoA and malonyl CoA are attached to acyl
carrier protein
2) Condensation - both are condensed by fatty acid synthase to
from acetoacetyl-ACP
3) Reduction - NADPH is oxidized to form hydroxybutyryl ACP
4) Dehydration - formation of double bond
+
5) Reduction - NADPH is source of e and H to form butyrylACP
Last four steps are repeated, each time with malonyl-ACP to elongate chain,
until palmitate is produced.
Overall reaction:
+
acetyl CoA + 7 malonyl CoA + 14 NADPH + 20 H ---> palmitate + 7CO2 + 14
+
NADP + 8 HS-CoA + 6 H2O
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Regulation of Fatty Acid Synthesis

Metabolism of fatty acids is under hormonal regulation by glucagons,
epinephrine, and insulin.
Fatty acid synthesis is maximal when carbohydrate and energy are
plentiful.
Important points of control are release of fatty acids from adipocytes and
regulation of carnitine acyltransferase I in the liver.
High insulin levels also stimulate formation of malonyl CoA, which
allosterically inhibits carnitine acyltransferase I  fatty acids remain in
cytosol and are not transported to mitochondria for oxidation.
Key regulatory enzyme is acetyl-CoA carboxylase (catalyzes first
committed step in fatty acid synthesis).
Insulin stimulates fatty acid synthesis and inhibits hydrolysis of stored
triacylglycerols.
Glucagon and epinephrine inhibit fatty acid synthesis (enzyme is
phosphorylated by protein kinase A; removal of phosphate group catalyzed
by protein phosphatase 2A).
Citrate is an allosteric activator, but its biological relevance has not been
established.
Fatty acyl CoA acts as an inhibitor.
Palmitoyl CoA and AMP are allosteric inhibitors.
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Synthesis of Eicosanoids

Precursors for eicosanoids are 20-carbon polyunsaturated fatty acids such
as arachidonate.
Part of inner leaflet of cell membrane.
There are two classes of eicosanoids:
1) prostaglandins and thromboxanes
Synthesized by enzyme cyclooxygenase
Localized molecules such as thromboxane A2, prostaglandins, prostacyclin
ae produced.
Thromboxane A2 leads to platelet aggregation and blood clots  reduced
blood flow in tissues.
Aspirin binds irreversibly to COX enzymes and prevents prostaglandin
synthesis.
2) leukotrienes
Produced by lipoxygenases.
Products were once called “slow-acting substances of anaphylaxis”,
responsible for fatal effects of some immunizations.
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Synthesis of Triacylglycerols and Glycerophospholipids
Most fatty acids are esterified as triacylglycerols or glycerophospholipids.
Intermediate molecule in synthesis of these two molecules is phosphatidic acid or
phosphatidate.
There are two pathways:
1) de novo – “from scratch”
2) salvage pathway - uses “old” pieces and parts to make new molecules
Synthesis of phosphatidate:
 Common intermediate in synthesis of phosphoglycerides and
triacylglycerols
 Formed from glycerol 3-phosphate and 2 acetyl CoA molecules
 Enzyme is glycerol phosphate acyltransferase
Synthesis of triacylglycerols and neutral phospholipids:
 Uses phosphatidate, which is dephosphorylated to produce 1,2diacylglycerol
If acetylated ---> triacylglyerol
If reacted with nucleotide derivative --> phosphatidylcholine or
phosphatidylethanolamine
Synthesis of acidic phospholipids:
 Uses phosphatidate and reacts it with CTP ---> CDP-diacylglycerol
 Addition of serine --> phosphatidylserine
 Addition of inositol ---> phosphatidylinositol

In mammals, phosphatidylserine and phosphatidylethanolamine can be
interconverted - base-exchange occurs in ER.
 Decarboxylation occurs in mitochondria and procaryotes
Synthesis of Sphingolipids
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All have C18 unsaturated alcohol (sphingosine) as structural backbone,
rather than glycerol
Palmitoyl CoA and serine condense ---> dehydrosphinganine --->
sphingosine
Acetylation of amino group of sphingosine ---> ceramide
Substitution of terminal hydroxyl group gives:

sphingomyelin -- addition of phosphatidylcholine

cerebroside -- substitute UDP-glucose or UDP-galactose

gangliosides -- substitute oligosaccharide
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Tay-Sachs disease = inherited disorder of ganglioside breakdown.

-N-acetylhexosaminidase, which removes
the terminal N-acetylgalactosamine residue from its ganglioside.
 One in 30 Jewish Americans of eastern European descent are carriers of a
defective allele.
 Can be diagnosed during fetal development by assaying amniotic fluid for
enzyme activity.
 Causes weakness, retarded psychomotor development, blindness by age
two, and death around age three.
Synthesis of Cholesterol
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Precursor of steroid hormones and bile salts.
Most cholesterol is synthesized in liver cells, although most animal cells can
synthesize it.
Starts with 3 molecules of acetyl CoA to form 3-hydroxy-3-methyl-glutaryl
CoA, which is reduced to mevalonate (C6) by HMG-CoA reductase (first
committed step of cholesterol synthesis)
Amount of cholesterol formation by liver and intestine is highly responsive to
cellular levels of cholesterol.
Enzyme HMG-CoA reductase is controlled in multiple ways:
1) Rate of enzyme synthesis is controlled by sterol regulatory element
(SRE); SRE inhibits mRNA production
2) Translation of reductase mRNA is inhibited by nonsterol metabolites
derived from mevalonate
3) Degradation of the enzyme occurs at high enzyme levels
4) Phosphorylation of enzyme
If enzyme is phosphorylated via glucagon pathway --> decreased activity-->
cholesterol synthesis ceases when ATP levels are low
If enzyme is dephosphorylated via insulin pathway --> increased activity

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Cells outside liver and intestine obtain cholesterol from blood instead of
synthesizing it de novo.
Steps in the uptake of cholesterol by LDL pathway:
1) apolipoprotein on surface of LDL particle binds to receptor on membrane of
nonhepatic cells
2) LDL-receptor complex internalized by endocytosis
3) vesicles formed fuse with lysosomes, which breaks apart protein part of
lipoprotein to amino acids and hydrolyzes cholesterol esters
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4) released unesterified cholesterol can be used for membrane biosynthesis or
be reesterified for storage
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Defects in LDL receptor lead to familial hypercholesterolemia (FH), in which
cholesterol and LDL levels are markedly elevated.
Result is deposition of cholesterol in tissues because of high levels of LDLcholesterol in blood
Heterozygotes suffer from atherosclerosis and increased risk of stroke
Homozygotes usually die in childhood from coronary artery disease
Disease is the result of an absence (homozygotes) or reduction
(heterozygotes) in number of LDL receptors.
LDL entry into liver and other cells is impaired.
Drug therapy can help heterozygotes
1) can inhibit intestinal absorption of bile salts (which promote absorption of
dietary cholesterol)
2) lovastatin - competitive inhibitor of HMG-CoA reductase ---> blocks
cholesterol synthesis
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CHAPTER 11 : Nucleotides, nucleic acids
Nucleic Acids Form Complementary Interactions
How is the potential for copying primary sequency made possible in nucleic acids?
The structure of nucleic acid makes it possible to form a duplex of two complementary
chains
The chains are complementary because each nucleic acid residue can form a unique
complementary interaction with another residue
In DNA, there are 4 types of residues (A, G, C, T) which form two complementary
interactions (A-T, G-C):
Complementary DNA Chains Form a Helical Structure
Two DNA chains that have a complementary sequence can form a double-stranded
helix:
Each of the two chains serves as a template for the formation of a new complementary
chain
As the original helix separates and new complementary chains are created, the result is
a copy of the original chain:
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The Flow of Genetic Information
This ability to copy the information in a DNA molecule enables a flow of information
in the cell
The stable information within DNA is copied to temporary messages of RNA which in
turn are used to create Protein:
A second flow of information occurs between cells, from one generation to the next
Nucleotides
Each residue in a nucleic acid is composed of three components:
a ribose sugar
a base
a phosphate group
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The Ribose Component
The ribose is a 5-membered ring
The atoms in the ring are numbered with a prime (') symbol, to distinguish them from
the numbered atoms in the base. Note that the sugars in DNA residues lack an oxygen on
the 2' carbon, so it is called deoxyribose
The Base Component
The second component of a nucleic acid residue is a planar, aromatic base
There are two kinds of bases: purines and pyrimidines
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The Phosphate Group
The third component is the phosphate group
By convention, the phosphate group of a residue is the one connected to the 5' carbon
of the ribose
At physiological pH, the phosphate group of each residue has a single negative charge,
which results in a large overall negative charge on the entire nucleic acid chain
In solution, the negative charges on the phosphates are compensated by nearby
positively-charged counter-ions or positively-charged side chains of nucleic-acid
binding proteins (lysines and arginines)
Nucleosides
The structure of the ribose and the base together, without the phosphate, is referred to
as a nucleoside
The four nucleosides in RNA are
adenosine
guanosine
cytidine
uridine
The four nucleosides in DNA are
deoxyadenosine
deoxyguanosine
dexoycytidine
deoxythymidine (thymidine)
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Nucleoside Structure
In nuclosides, the base is attached to the ribose at the C1' carbon in a -glycosidic
linkage:
This is the bond from the anomeric carbon that comes up out of the plane of a sugar
In nucleosides with purine bases, the C1' carbon connects to
the N9 atom of the purine
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In nucleosides with pyrimidine bases, the C1'carbon connects to the N1 atom of the
pyrimidine
Nucleotides
When a nucleoside is joined to one or more phosphates, it is referred to as a nucleotide
In DNA, phosphates can be attached at the 5' and the 3' carbons
In RNA, phosphates can be attached at the 5', 3' and 2' carbons
A nucleoside with a group attached to the 5' end is a nucleoside 5'-phosphate
For example, the nucleotide ATP has a triphosphate group at its 5' end, so it is called 5'ATP or adenosine 5'-triphosphate or adenylate
The type of nucleoside and the number and position of the phosphate groups affect the
naming of the nucleotide
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The directionality of nucleic acid is shown in a 2D structural
representation of a tetranucleotide of DNA: pdApdGpdTpdC, or more
commonly, just AGTC The individual residues are linked
together by 3'-5' phosphodiester covalent bonds
The 'backbone' of the structure consists of the phosphoryl groups
and the deoxyribose moieties
Note the unit negative charge for each residue in the chain, at normal physiological pH
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The DNA Helix
As mentioned previously, two nucleic acid chains with a complementary sequence can
associate to form a double helix
Depending upon the type of nucleic acid and the conditions, two types of helices are
commonly found: the A-form helix and the B-form helix
Two complementary strands of DNA can adopt either the A-form or the B-form helix,
depending upon the conditions
One DNA strand and one RNA strand or two RNA strands will associate only in the
Aform geometry
The B-Form Double Helix
Under normal physiological conditions, DNA is found in the B-form double helix
The double helix is a right-handed helix, with the two strands running in antiparallel
orientation:
There are 10 residues per turn, with a pitch of 34A
The diameter of the helix is 20A
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The Helix is Stabilized by Base Pairs
The flat nucleotide bases lie perpendicular to the helix axis and point inward toward
the center
Two bases on opposite strands form a complementary interaction called a base pair
The base pairing results from hydrogen bonds that form at the base edges
Adenine forms two h-bonds with thymine, guanine forms three h-bonds with cytosine
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Chemical Structure of Double-Stranded DNA
The 2D structural representation of a doublestranded
sequence of DNA shows the two strands running in opposite or antiparallel
orientations The negatively-charged phosphate groups are on the outside and the bases are
hydrogen bonded in the interior. The negatively-charged phosphate groups are on the
outside and the bases are hydrogen bonded in the interior. Note again that in the actual 3D
structures, the planes of the bases are oriented mostly perpendicular to the plane of the
ribose sugars in the backbone
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