Biochemistry
Extended Syllabus in English
Prepared by: Radovan Hynek and Olga Valentová
Lecture 1
INTRODUCTION - ORGANIZATION OF LIVING
SYSTEMS
Biochemistry describes living organisms on molecular level by chemical approaches.
Each organism and even the smallest cells consist of thousands inorganic and organic
compounds, the letter varying from small molecules to large biopolymers.
All biological processes like vision, digestion, motion, immunity, disease and even thinking
are based on the action and interaction of these molecules. Thus the knowledge of chemical
structure as well as their biological function is necessary.
The respective roles of chemistry and biology in achieving the goals of biochemistry are
readily apparent. Of the same importance in understanding the processes in living system is
the energy flow (bioenergetics) as some molecular events in the cell require energy while
others release energy.
Biochemistry is devided into two levels:
1. Conformational: discovering the chemical composition of organisms, structure and
three dimensional organization of the molecules, organization of supramolecular
structure, relation between structure and function of the molecules
2. Informational: describes metabolism, bioenergetics and physiological processes on
molecular level
Biochemistry is divided into many sub-domains according to the field of interest:
Molecular genetics studying the transfer of genetic information on molecular level
(compare to Mendelian genetic)
Pathobiochemistry - biochemistry of diseases
Clinical biochemistry – analysis of body fluids as a part of desease diagnostics
Biotechnology – studies technological applications that use biological systems or their parts.
Xenobiochemistry (farmacological biochemistry) - deals with the fate of drugs and toxins in
organisms.
Biophysical chemistry – uses the approaches of physical chemistry to solve biological
problems
Bioorganic chemistry – study of biologically active organic compounds
COMPONENTS AND ORGANIZATION OF LIVING ORGANISMS
Living organisms differ from inanimate objects in many aspects: they are very complex with
high degree of organization, able to extract energy from nutrients, regulate their functions,
actively respond to the changes of their environment, grow and reproduce.
From elements to biomolecules
Bulk elements essential for life: C,H,O,N,P and S make up to 92%of the dry weight of living
objects.
Elements in trace quantities essential for life: Ca, Na, K, Mg, Fe, I and Cl
Other trace elements: As, B, Mo, Cu, Zn etc.
Table I. Molecular composition of different types of living organisms
Component Rel. mol. Amount in organism (g/100 g) Number molecular
weight
species in bacterial
cells
human plant
bacteria
water
proteins
DNA
RNA
saccharides
lipids
Other organic
compounds
Inorganic
compounds
18
10 -106
60
18
75
4
70
15
1
3 000
>106
4.104-106
<1
1,5
<1
1
1
6
1
1 000
102-106
0,5
16
2
250
750-1 500
16
1
2
50
100-500
1
1
2
500
Approx.
60
3
2
1
15-20
4
Water is more than solvent in living systems
All components mentioned in Table I will be discussed in more details in the following
chapters except water. It is worthwhile to emphasise its unique role in biological systems
here:
- water represents about 60 to 70 % of the fresh weight of living
organisms
- all reactions occurring in living organisms are performed in water
solution
- water is a reactant or product of many biochemical reactions
- photolytic cleavage of water molecule is one of the principal reaction
on the Earth
Processes running in living organisms are base on non-covalent
interactions
Table II Overview of non-covalent interactions in living systems
Type of interaction
Example
Hydrogen bridges:
water (ice)
Peptide bond
electrostat. interactions
ion-ion
Interaction of
permanent dipols
-O-H...O=
Energy
(kJ/mol)
17
=N-H...O=C
-COO-...+H3N-
15
20-30
|
|
Cδ+=Oδ-...Cδ+=Oδ|
|
two aliphatic carbons
2
0,11
two aromatic rings
6
two methyl groups
1,2
Londonovy dispersní
interakce
Stacking interactions??
hydrofobic interaction
Molecular recognition is the result of an exact fit between the surfaces of two molecules.
Complementary molecules form a complex that displays certain biological activity (enzymesubstrate, hormone-receptor, antibody-antigene etc). This phenomenon is the basis of all
processes in living organisms.
Organization of living organisms
Small molecules – not very many (hundreds) metabolites or monomers from which the
biopolymers are built (amino acids, monosacharides, purine and pyrimidine bases, fatty
acids).
Biopolymers – much more diverse than small molecules, thounds of different biopolymers in
one cell. They are built from monomers: proteins from aminoacids, nucleic acids from
monosaccharide ribose or deoxyribose, nucleic base and phosphate group, polysaccharides are
composed from monosaccharides.
Supramolecular assemblies – clusters composed from thousands of biopolymer molecules
highly organized ( cytoskeleton, ribosomes, chromatine). The exception are the biological
membranes composed from phospholipids.
Cytoskeleton - three dimensional fibrous matrix extended
throughout inside of the eukaryotic cell, gives the shape
to the cell, enables the movement and guides the internal
movement of organelles. The long fibers of the
cytoskeleton are polymers of subunits. The fibres are
primarily composed from proteins and are of three types:
microtubules composed from protein tubulin and
microfilamnets composed of actine and finally
The eukaryotic cytoskeleton. actin filaments
are shown in red, microtubules in green, and
the nuclei are in blue.
intermediate filaments varies from cell to cellThe primary types of fibers comprising the
cytoskeleton are microfilaments, microtubules, and intermediate filaments.
Biological membranes
The cell membrane consists primarily of a thin layer of amphipatic phospholipids which
spontaneously arrange so that the hydrophobic "tail" regions are shielded from the
surrounding polar fluid, causing the more hydrophilic "head" regions to associate with the
cytosolic and extracellular faces of the resulting bilayer. This forms a continuous, spherical
lipid bilayer.
The arrangement of hydrophilic heads and hydrophobic tails of the lipid bilayer prevent polar
solutes (e.g. amino acids, nucleic acids, carbohydrates, proteins, and ions) from diffusing
across the membrane, but generally allows for the passive diffusion of hydrophobic
molecules. This affords the cell the ability to control the movement of these substances via
transmembrane protein complexes such as pores and gates. Membranes serve diverse
functions in eukaryotic and prokaryotic cells. One important role is to regulate the movement
of materials into and out of cells. The phospholipid bilayer structure (fluid mosaic model)
with specific membrane proteins accounts for the selective permeability of the membrane and
passive and active transport mechanisms. Transmembrane proteins serve also as receptors of
various signals .. Beside these integral mebrane proteins there are many proteins associated
with the mebrane surface (peripheral membrane proteins) with various functions.
Viruses – consist of DNA or RNA molecule in the protein envelope. Viruses cannot exist
independently and are usually not considered as a life-form , in this intention they can be
considered as supramolecular assemblies as well.
After suplamolecular assemblies the higher level of organization is the fundamental unit of
life, the cell. Generally living organisms are divided to two basic groups: prokaryotes and
eukaryotes.
Prokaryotes
Simple, unicellular organisms, mainly
bacteria and blue/green algae with neither
the distinct nucleus or intracellular
compartmentalization. They are the most
abundant organisms on the earth.
Eukaryotes
This class of living organisms includes
animals, plants and fungi, protozoan, yeast
and some algae. The comlex eukaryotic cells
are much larger than prokaryotic, with
diameter ranging between 10 to 100 µm. They
are surrounded by plasma membrane and
except the animal cell also with cell wall.
Inner space of the cell is compartmentalized to
organelles,
membrane enclosed packages of organized
molecules that perform specialized functions. Eukaryotic cells are of very different shape and
size.
Plant cell
Lecture 2
AMINO ACIDS AND PEPTIDES
The amino acids are the building blocks for proteins - nearly all proteins studied are made
from the twenty "standard" amino acids we will look at now. All of the standard amino acids
are alpha amino acids (except for proline, an imino acid). That is they have an amino group
alpha to the carboxyl group (they are 2-amino acids). Amino acids share the basic structure
below and occur in two optical forms:
COO-
COO-
⏐
⏐
D: H⎯ C⎯NH3+
L:
NH3+ ⎯ C⎯H
⏐
⏐
R
R
Only amino acids of L forms (coded in DNA) are building blocks of proteins
L α-Amino Acids Found in Proteins
Side chains (indicated in blue in the diagram).
orange area are nonpolar and hydrophobic
magenta box are acidic ("carboxy" group in the side chain).
blue box are basic ("basic" group in the side chain).
Amino Acid Classifications
Each of the 20 α-amino acids found in proteins can be distinguished by the R-group
substitution on the α-carbon atom. There are two broad classes of amino acids based upon
whether the R-group is hydrophobic or hydrophilic.
The hydrophobic amino acids tend to repel the aqueous environment and, therefore, reside
predominantly in the interior of proteins. This class of amino acids does not ionize nor
participate in the formation of H-bonds. The hydrophilic amino acids tend to interact with the
aqueous environment, are often involved in the formation of H-bonds and are predominantly
found on the exterior surfaces proteins or in the reactive centers of enzymes.
Acid-Base Properties of the Amino Acids
The α-COOH and α-NH2 groups in amino acids are capable of ionizing (as are the acidic and
basic R-groups of the amino acids). As a result of their ionizability the following ionic
equilibrium reactions may be written:
R-COOH <——> R-COO– + H+
R-NH3+ <——> R-NH2 + H+
The equilibrium reactions, as written, demonstrate that amino acids contain at least two
weakly acidic groups. However, the carboxyl group is a far stronger acid than the amino
group. At physiological pH (around 7.4) the carboxyl group will be unprotonated and the
amino group will be protonated. An amino acid with no ionizable R-group would be
electrically neutral at this pH. This species is termed a zwitterion.
KA =
[ ][ ]
H + A-
[ HA]
[H ] = K
[ HA]
+
A
[ ]
A-
[A ]
+ log
−
pH = pK A
[ HA]
Like typical organic acids, the acidic strength of the carboxyl, amino and ionizable R-groups
in amino acids can be defined by the association constant, Ka or more commonly the negative
logrithm of Ka, the pKa. The net charge (the algebraic sum of all the charged groups present)
of any amino acid, peptide or protein, will depend upon the pH of the surrounding aqueous
environment. As the pH of a solution of an amino acid or protein changes so too does the net
charge. This phenomenon can be observed during the titration of any amino acid or protein.
When the net charge of an amino acid or protein is zero the pH will be equivalent to the
isoelectric point: pI.
At neutral pH (pH =7) both the acid and amine groups will be ionized to give the so-called
zwitterion form. Note that there is no pH at which the amino acid structure will have no
ionized groups! Note the titration behavior of amino acids, and be able to draw the structure
for an amino acid at each point in the curve.
Functional Significance of Amino Acid R-Groups
In solution it is the nature of the amino acid R-groups that dictate structure-function
relationships of peptides and proteins. The hydrophobic amino acids will generally be
encountered in the interior of proteins shielded from direct contact with water. Conversely,
the hydrophilic amino acids are generally found on the exterior of proteins as well as in the
active centers of enzymatically active proteins. Indeed, it is the very nature of certain amino
acid R-groups that allow enzyme reactions to occur.
The imidazole ring of histidine allows it to act as either a proton donor or acceptor at
physiological pH. Hence, it is frequently found in the reactive center of enzymes. Equally
important is the ability of histidines in hemoglobin to buffer the H+ ions from carbonic acid
ionization in red blood cells. It is this property of hemoglobin that allows it to exchange O2
and CO2 at the tissues or lungs, respectively.
The primary alcohol of serine and threonine as well as the thiol (–SH) of cysteine allow these
amino acids to act as nucleophiles during enzymatic catalysis. Additionally, the thiol of
cysteine is able to form a disulfide bond with other cysteines:
Cysteine-SH + HS-Cysteine <——> Cysteine-S-S-Cysteine
This simple disulfide is identified as cystine. The formation of disulfide bonds between
cysteines present within proteins is important to the formation of active structural domains in
a large number of proteins. Disulfide bonding between cysteines in different polypeptide
chains of oligomeric proteins plays a crucial role in ordering the structure of complex
proteins, e.g. the insulin receptor.
• Nonpolar side chains: these will tend to be found on interior of protein, except that glycine
and alanine are so small that they can fit into interior or on surface. Compare these amino
acids: note how these side chains build in size from gly (glycine), ala (alanine), val (valine), to
leu (leucine), then have two which have about same size but different shapes: ile (isoleucine)
and met (methionine - met has a nearly identical shape to the linear analogue of leucine,
norleucine). Met of course also has possibility of liganding metal ions through sulfur. Next
have phe (phenylalanine) and trp (tryptophan). These are aromatic, which enables stacking
interactions with other aromatic groups as well as being very hydrophobic. Trp also has an
amine group which needs to form a hydrogen bond. Thus trp is often found with the -NH at
the surface but with the remainder in a hydrophobic cleft. If trp is interior it will generally
hydrogen bond with another functional group. Finally pro (proline) is also hydrophobic, but
its main characteristic of interest is its tendency to put a near right angle in the direction of a
peptide chain. It thus generally disrupts particular structural elements of proteins. As such it is
often near the surface, since it forces structural elements to turn at the surface (defining the
surface).*
Uncharged Polar side chains: These side chains will generally occur on the surfaces of
proteins because of their polarity and hydrogen-bonding characteristics. If they occur on the
interior they must generally H-bond with other interior functional groups. The definition of
"uncharged" is based on a pH of 7. There are four side-chains, ser (serine), thr (threonine), asn
(asparagine), and gln (glutamine), which are neutral under all conditions of pH. (Note that asn
and gln are simply the amide forms of asp and glu. It is thus often difficult to determine
whether a given residue was a asp or asn etc. in chemical analysis of peptides, since the
treatment breaking peptide bonds also will generally break the amide bonds of asn and gln.)
Tyr (tyrosine) and cys (cysteine) are uncharged at pH 7, but both ionize at higher pH's
(respective pKa's = 9.5-10.9 & 8.3-8.6). Finally, his (histidine - imidazolium grp), has a pKa of
6.4-7.0 and is thus partially charged (positive) at pH 7, and will be charged at low pH's.
•
• Charged Polar side chains: These four side-chains will have very strong tendencies to be
on the surface - it costs a great deal of energy to bury an ionic charge in a non-polar interior!
It turns out that the sum of the acidic groups in a protein, asp (aspartate) + glu (glutamate), is
usually equal to the sum of the sum of the basic groups, lys (lysine - amino grp) + arg
(arginine - guanidinium grp). This is expected since we want a net neutral particle at its
operating pH (usually around pH 7)
Peptides and Amino Acid Chemistry
• Peptide bond formation: Peptide bond is simply an amide bond between the alpha
carboxyl and amino groups of amino acids. If we write the reacting groups in their unionized
(acid and amine) forms, then we can see the reaction takes place with the loss of the elements
of water, via an attack of the lone-pair electrons of the amine on the carbonyl carbon of the
carboxyl group:
•
•
The peptide bond is formed with the elimination of water, giving a planar bond
between the carboxyl carbon and the amino nitrogen. This is due to the partial double bond
character on the amide/peptide bond as seen in the shorter bond length (0.133 nm vs. 0.146
nm). This bond is nearly always trans in proteins due to steric interactions of the amide
hydrogen and oxygen, except for proline.
Examples of peptides
Glutathione (abbreviated GSH) is a tripeptide composed of glutamate, cysteine and glycine
that has numerous important functions within cells. Glutathione serves as a reductant; is
conjugated to drugs to make them more water soluble; is involved in amino acid transport
across cell membranes (the γ-glutamyl cycle); is a substrate for the peptidoleukotrienes;
serves as a cofactor for some enzymatic reactions and as an aid in the rearrangement of
protein disulfide bonds.
Synthesis of Glutathione
(GSH)
Structure of GSSG
The role of GSH as a reductant is extremely important particularly in the highly oxidizing
environment of the erythrocyte. The sulfhydryl of GSH can be used to reduce peroxides
formed during oxygen transport. The resulting oxidized form of GSH consists of two
molecules disulfide bonded together (abbreviated GSSG). The enzyme glutathione reductase
utilizes NADPH as a cofactor to reduce GSSG back to two moles of GSH. Hence, the pentose
phosphate pathway is an extremely important pathway of erythrocytes for the continuing
production of the NADPH needed by glutathione reductase. In fact as much as 10% of
glucose consumption, by erythrocytes, may be mediated by the pentose phosphate pathway.
Other biologically aktive peptides: insulin, oxytocin, vasopresin, endorfins etc.
Lecture 3
PROTEIN STRUCTURE AND FUNCTION
Proteins are commonly large (MW > 6,000), globular molecules serving many functions
Levels of protein structure:
•
Primary structure : the linear order or sequence of peptide bonded amino acid
residues, beginning at the N-terminus. (Characteristic bond type: covalent.)
•
Secondary structure: the steric relations of residues nearby in the primary structure
which give rise to local regularities of conformation. These structures are maintained
by hydrogen bonds between peptide bond carbonyl oxygens and amide hydrogens.
The major secondary structural elements are the alpha helix and the beta strand.
(Characteristic bond type: hydrogen.)
•
Tertiary structure (3°): the steric relations of residues distant in the primary
sequence; the overall folding pattern of a single covalently linked molecule.
(Characteristic bond type: hydrophobic; others: hydrogen, ion-pair, van der Waals,
disulfide.)
o
•
Domains: independent folding regions within a protein. The group/pattern of
secondary structures forming a Domain's tertiary structure is called a Fold.
(Characteristic bond type: hydrophobic; others: hydrogen, ion-pair, van der
Waals.)
Quarternary structure: the association of two or more independent proteins via noncovalent forces to give a multimeric protein. The individual peptide units of this
protein are referred to as subunits, and they may be identical or different from one
another. (Characteristic bond type: hydrophobic; others: hydrogen, ion-pair, van der
Waals.)
Levels of protein structure:
Protein Folding
Primary structure specifies tertiary (& therefore quaternary) structure. This is known from in
vitro denaturation/renaturation studies of small proteins.
•
•
Denaturation means to unfold to non-functional state, often achieve a "random coil"
in solution.
o Denatruration is cooperative, that is takes place all at once as seen in
denaturation curves
Renaturation means to return to the properly folded, natural, and functional state.)
The classic study involved Ribonuclease: Reduce (break) -S-S- bonds, denature with urea to
random coil. Now can renature by gently removing denaturant (urea) and oxidize -S-S- bonds.
X-ray diffraction image is also the same! Note - no gremlins, no magic, done in "test tube."
Other small proteins, such as Myoglobin and proinsulin, fold up spontaneously in the same
manner as Ribonuclease. However, insulin fails to fold correctly, since a peptide essential to
folding has been cleaved off.
Accessory Folding Proteins. The ribonuclease renaturation-type experiment has not been
repeated with large proteins, which seem to require the participation of "folding catalysts," to
aid their folding: the Chaperones.
Chaperones
•
•
•
Renaturation-type experiment has not been repeated with large proteins.
Many proteins are aided in folding process by "folding catalysts." These so-called
chaperones appear to stabilize unfolded conformations, allowing time to find correct
folding pattern.
Some chaperones require ATP energy to function. A number of different type:
o The so-called Heat-shock proteins (Hsp70) are a family of chaperons
The Chaperonins (Hsp60 or GroEL and Hsp10 or GroES) are barrel-like proteins providing an
internal folding environment. GroEL is large enough to accomodate a protein with >600
residues.
Thermodynamics notes to protein folding
Let's look at folding in another way: You might guess a protein would fold to lowest free
energy conformation. Problem: is there time? ("Levinthal's Paradox", formulated by Cyrus
Levinthal in 1968) Stryer calculation (very conservative): Assume 100 aa residue protein with
3 possible conformations/residue; then get 3100or 5 x 1047 possible conformations. If search at
a rate of one structure/10-13sec then get (5 x 1047)(10-13)= 5 x 1034 sec or 1.6 x 1027 years to
search (and thus to fold protein).
Obviously from these calculations not searching all possible conformations (or we have the
process wrong!), so cannot say protein achieves the lowest global free energy, but rather a
local free energy minima and by compaction, they may unfold and try other combinations
until stable associations result.
Protein functions
Proteins have numerous functions – e.g.: They serve as enzymatic catalysts, are used as
transport molecules (hemoglobin transports oxygen) and storage molecules (iron is stored
in the liver as a complex with the protein ferritin); they are used in movement (proteins
are the major component of muscles); they are needed for mechanical support (skin and
bone contain collagen-a fibrous protein); they mediate cell responses (rhodopsin is a
protein in the eye which is used for vision); antibody proteins are needed for immune
protection; control of growth and cell differentiation uses proteins (hormones) etc.
Demonstration of protein functions on examples:
Myoglobin
Myoglobin is a 153 residue globular protein in the globin family. Eight alpha helices form its
single domain (myoglobin fold) tertiary structure; about 80% alpha helix (high for globular
proteins). Interior almost exclusively hydrophobic residues, with water excluded from
interior. Surface has mix of hydrophobic and hydrophilic residues, with ionizable groups on
surface.
Myoglobin functions to store and facilitate the diffusion of oxygen in muscle. Oxygen binds
to a heme {Fe (II)-protoporphyrin IX} prosthetic group. Four of iron's six ligands are to heme
nitrogens, with a fifth to a histidine nitrogen. The final ligand bond goes to oxygen. Breathing
motions (see below) are necessary to allow the exchange of oxygen, since the heme is in a
closed pocket.
Protein Dynamics
"Breathing" motions:
•
•
•
Atomic fluctuations (10-15- 10-11 sec; 0.001 - 0.1 nanometer) Myoglobin example
[overhead v&v 8.9, 8.10]
Collective motions of covalently linked atoms, from aa R-groups to domains (10-12 10-3 sec; 0.001 - >0.5 nanometer)
Triggered conformational changes: in response to ligand binding, covalent
modification etc.
How do we know about the mobility of protein structures?
•
•
•
•
X-ray diffraction studies of proteins with and without ligand bound
NMR (phe, his ring protons/carbons show up on edges of signal envelope)
H-exchange
Antibody binding: make antibodies to normally interior aa residues, over time protein
ppt forms as interior groups momentarily exposed.
Oxygen Binding
Myoglobin
Let's look at binding in terms of saturation, Y, where if Y = 1 every site of every Myoglobin
is occupied by an oxygen molecule (thus if Y = 0.5, then 50% of the myoglobin are binding
oxygen and 50% are "empty"). Mb/Hb binding curve :
Reviewing the curve in terms of saturation, Y, if Y = 1 then every site of every Myoglobin is
occupied by an oxygen molecule (thus if Y = 0.5, then 50% of the myoglobin are binding
oxygen and 50% are "empty").
Can describe binding as dissociation equilibrium,then:
MbO2
for saturation. Substituting,
Mb + O2 ;
&
, the equation of a hyperbola. If expressed as
pressures, then
where P50 = pO2 @ 50% saturation. Note that the binding curve
for Mb is indeed hyperbolic in shape.
Hemoglobin
Hemoglobin is an alpha-alpha-beta-beta oligomeric protein: its quaternary structure consists
of a tetramer of myoglobin like subunits. The two types of chain are slightly shorter than
myoglobin chains (alpha= 141 aa residues, beta= 146 aa residues). There are extensive
contacts between an alpha and a beta subunit to give a dimer. The dimers have additional
contacts to give the tetramer. Oxygen binding results in a change of conformation in Hb. The
change of conformation affects the binding of oxygen {oxygen binding is reduced in the
"blue" form due to steric hindrance between the oxygen and the heme}.
What about Hb oxygen binding? Obviously more complex. The sigmoid shape (s-shape) of
the curve indicates cooperativity. That is, if one site binds, another is more likely to as well (it
cooperates with the first site).
Lecture 4
ENZYMES: COMMON CHARACTERISTIC
AND CLASSIFICATION
The enzymes are catalysts for biochemical reactions in living organisms. They direct and
regulate the thousands of reactions providing for energy transformation, synthesis and
metabolic degradation.
As catalysts generally, the enzymes lower the activation energy of the reaction.
Compared to chemical catalyst they have some unique features: enzyme work under mild,
physiological conditions with high efficiency and specificity and their activity can be
regulated.
Basic terms:
substrate – reactant of the enzyme catalyzed reaction,
product of the catalyzed reaction
active site – the region that contains catalytic groups,
binds the substrate, and then carries out the reaction
catalytic site (catalytic groups) - groups of atoms (i.e
atoms from the side chains of amino acids or cofactors or
metal ions) in the enzyme molecule which are directly
involved in the chemical change of the substrate.
Tab Reaction rates of the decomposition of hydrogen peroxide in presence of different
catalysts
Catalyst
non
HBr
Fe(OH)2-triethylen
tetraamine
Catalase
Reaction rate
(mol.l-1.s-1)
10-8
10-4
103
Ea (kJ.mol-1)
107
8,4
71,1
50,2
29,3
Basically the enzymes are proteins , but can contain also a nonproteinaceous part - cofactors
which are necessary for their function. Cofactors are either covalently bound to peptide chain
- prosthetic group ( FAD, lipoamide, biotin) or noncovalently associated with protein –
coenzyme (NAD+, CoA, ATP).
Enzyme nomenclature – classification of enzymes
A systematic scheme for classification of enzymes was established in 1972 by the
International Union of Biochemistry. Each enzyme is designated by EC number with four
numbers indicating class (x), subclass (y), subsubclass (z) and ordinal number
EC x.y.z.i
Enzymes are divided into six classes according to the type of catalyzed reaction:
Class
EC 1
Oxidoreductases
EC 2
Transferases
EC 3
Hydrolases
EC 4
Lyases
EC 5
Isomerases
EC 6
Ligases
Reaction catalyzed
Typical reaction
Enzyme example(s)
with trivial name
Catalyze oxidation/reduction reactions;
transfer of H and O atoms or electrons
from one substance to another
AH + B → A + BH A+ B → A + B-
Dehydrogenase,
oxidase
Transfer of a functional group from one
substance to another. The group may be
methyl-, acyl-, amino- or phosphate group
AB + C → A + BC
Transaminase, kinase
Hydrolysis of substrate
AB + H2O → AOH +
BH
Lipase, amylase,
peptidase
Non-hydrolytic addition or removal of
groups from substrates. C-C, C-N, C-O or
C-S bonds may be cleaved
RCOCOOH → RCOH
+ CO2 or [x-A-B-Y]
→ [A=B + X-Y]
Decarboxylase
Intramolecule rearrangement, i.e.
isomerization changes within a single
molecule
AB → BA
Isomerase, mutase
Join together two molecules by synthesis
of new C-O, C-S, C-N or C-C bonds with
simultaneous breakdown of ATP
X + Y+ ATP → XY +
ADP + Pi
Synthetase
Subclasses represent the type of substrate and subsubcalss the special features of the enzyme
(i.e. acceptor).
Cofactors
NAD+, FAD, CoA,
Lecture 5
ENZYME KINETICS
Study the rate of the conversion of substrate to products (S → P):
Michaelis-Menten model for one substrate reaction:
E+S
k+1
k-1
k+2
ES (k ) E + P
Reversible substrate
binding
-2
Irreversible
product
formation
Initial reaction rate depends on the substrate and initial enzyme concentration
vo =
vo =
k 2 . [ Eo ] . [ S ]
k - 1 + k2
+ [S ]
k1
k 2 . [ Eo ] . [ S ]
KM + [S ]
KM = Michaelis konstant – equal to
substráte concentration at which the
velocity of the reaction is half maximal,
has a dimension of a concentration,
Michaelis-Menten equation
vo =
V lim. [S ]
KM + [ S ]
Vlim = k2 . [Eo]
Maud Menten
V lim
= k 2 = kcat
[ Eo ]
kcat- turnover number (molecular activity ) of the enzyme - maximum number of molecules
of the substráte that could be converted to produkt by one molekule of enzymr in one second
Substrate binding
"Lock and key" model
Enzymes are very specific, and it was suggested by Emil Fischer in 1894 that this was
because both the enzyme and the substrate possess specific complementary geometric shapes
that fit exactly into one another. This is often referred to as "the lock and key" model.
However, while this model explains enzyme specificity, it fails to explain the stabilization of
the transition state that enzymes achieve. The "lock and key" model has proven inaccurate,
and the induced fit model is the most currently accepted enzyme-substrate-coenzyme figure.
Induced fit model
In 1958, Daniel Koshland suggested a modification to
the lock and key model: since enzymes are rather
flexible structures, the presence of substrate induces
conformational changes in the protein molecule
especially in the active site region, the active site is
continually reshaped by interactions with the substrate
as the substrate interacts with the enzyme. The region
of active side not only recognizes the substrate
molecule but also orients it in such a way that facilitates
the catalytic reaction.
Stabilization of the transition state
Mechanism of catalysis
( example: acid catalysis by serine proteases)
Factors affecting enzyme acitivity
Enzyme inhibition
Inhibitors are specific agents that interfere with binding of a substrate to the active site or
with conversion of the enzyme-substrate complex into products:
Immobilized enzymes
An enzyme fixed by physical or chemical means to a solid support–e.g., a bead or gel to confine a
reaction of interest to a particular site. Immobilized enzyme preparation can be used repeatedly and
continuously. They are used in biotechnologies.
Immobilization of
enzymes
Binding to the
matrix
Adsorption
Covalent bond
Entrapment
In the gel matrix
encapsulation
Effect of immobilization on the enzyme:
1. Inactivation by reactants or products of immobiliztion reaction
2. Conditions of the immobilization reaction
3. binding forces or bonds fix the enzyme molekule in an inactiv or not fylly aktive
conformation
4. covalent bond formed with functional residuem of the aktive site
5. Orientation of the enzyme molekule limits the substrate Access to aktive site
6. influence of the funcional groups of the matrix (e.g. charged, hydrophobic).
Influence of the charge of matrix on the pH optimum of the immmobilized enzyme a.
positively chrged matrix b/ native enzyme c. negatively charged matrix
Aplication of enzymes in technology
Food and non-food industry
Clinical biochemistry (diagnostics and determination of analytes)
Pharmaceuticals
Research (genetic engineering etc.)
Enzymes used:
Hydrolases (80%) / glycosidases and proteases, lipases
Isomerases (12% - glucose isomerase)
Oxidoreductases and others (5%)
Sources of enzymes for technology
Microbial enzymes, bacterial and fungi, extremophiles, recombinant enzymes
Animal and plant – less than 5% , limitations
Lecture 6
NUCLEIC ACID STRUCTURE
Nucleic acids - deoxyribonucleic acid (DNA), ribonucleic acid (RNA) are biopolymers built
form monomer units nucleotides
Nucleotide (nucleoside mono, di, tri – phosphate)
nucleoside
Sugar – ribose (RNA), 2’- deoxyribose (DNA)
Nitrogenous base :
– purine - adenine (A), guanine (G)
– pyrimidine – cytosine (C), thymine (T), uracil (U) (replaces thymine in RNA)
–
adenine
guanine
cytosin
e
thymine
uracil
Character of nucleobases :
Weak bases, planar,
keto-enol tautomerism
spectral characteristics - UV
Nucleotide functions
- Building blocks of nucleic acids
- intracellular energy transfer
- donor of phosphate group
- activation of intermediates for biosynthesis (UDP glucose, CDP choline)
- structural components of cofactors,
vitamins (NAD(P)+. FAD, PAPS,
CoA, cobalamine)
- Regulation (second messengers,
neuromodulators - cAMP, cGMP)
- Therapeutics (antivirotics )
Cyclic AMP
3’-azido-2’,3’ dideoxythymidin
(AZT )
DNA structure
Primary structure – nucleotides linked with 3’,5’-phosphodiester bond in the direction
from5’ to 3’ end. Invariant backbone is formed by alternating phosphate and deoxyribose
units. Heterocyclic bases linked to the covalent backbone by N-glycosidic bonds on the 1’ C
atom of the monosaccharide.
The primary structure is recorded by using the letter symbols - the following structure then as
… TCAG…
Secondary structure - double helix, antiparalel strands, base pairing according to
complementarity of bases , fixed with hydrogen bonds
In a DNA molecule, the two strands are not parallel, but intertwined with each other. Each
strand looks like a helix. The two strands form a "double helix" structure, which was first
discovered by James D. Watson and Francis Crick in 1953. In this structure, also known as
the B form, the helix makes a turn every 3.4 nm, and the distance between two neighboring
base pairs is 0.34 nm. Hence, there are about 10 pairs per turn. The intertwined strands make
two grooves of different widths, referred to as the major groove and the minor groove,
which may facilitate binding with specific proteins.
Tertiary structure - supercoiled DNA
-
circular chromosome in prokaryotes,
linear chromosome in eukaryots – nucleosome, histones
RNA structure
Single stranded with ribose and uracil instead of thymine in the primary structure
Partial secondary structure - double helices, bulges, loops, hairpin turns formed within the
single strand
rRNA - ribosomal (~80% of RNAs in the
organisms), forms ribosomes together with
proteins
mRNA - messenger (~15% of RNAs), linear
molecule, transcribed from DNA template,
translated to proteins
tRNA – transfer RNA (~5%), the smallest
molecules of approx 75 to 95 nucleotides,
contain modified purine and pyrimidine
bases, displays the three-dimensional structure
with double helices and loops. Carries an
anticodon (three nucleotide bases) and a
specific amino acid which is tranferred to the growing polypeptide chain in proteosynthesis .
The identity of the amino acid is determined by the sequence of nucleotides in the anticodon.
Lecture 7
STORAGE AND UTILIZATION OF GENETIC
INFORMATION
Genetic information flow:
1) From DNA to DNA during its transmission
from generation to generation - replication
2) From DNA to Protein during its phenotypic
expression in an organism.
Transcription: DNA to RNA. Occassionally,
genetic information flows from RNA to DNA
(reverse transcription).
Translation: RNA to protein (irreversible).
Replication of DNA
When cell is dividing, complete genetic
information has to be copied an given to
daughter cell. Each chromosome hast to be
duplicated, process is called replication.
This process is semiconservative : two original strands are separated and each acts as a
template for the synthesis of a new strand on the principle of the complementarity of bases
A=T and G≡C.
Phases of the replication: Initiation, elongation, termination and processing
Replication fork, replication bubble
Topoisomerase - removes supercoils ahead of the replication fork
Helicase – separates two nucleic acid strands
DNA primase - synthesizes a short RNA segment (called a primer) complementary to a
ssDNA template. DNA polymerases cannot initiate the synthesis of a DNA strand without an
initial primer with free 3’OH group.
DNA polymerase - key enzyme: dNTP + (DNA)n → (DNA)n+1
+ PPi, can synthetize
polynucleotide chin only in the direction 5‘ → 3‘
Leading strand - continuously synthetized strand
Lagging strand - discontinuously synthetized short oligonucleotides
Okazaki fragments
DNA ligase - forming bonds between Okazakiho fragments to finish the synthesais of lagging
strand
DNA sequencing - determination of primary structure of DNA
Polymerase chain reaction - a technique to amplify a single or few copies of a piece of DNA
across several orders of magnitude, generating thousands to millions of copies of a particular
DNA sequence.
Gene expression
Genetic information encoded in the sequence of nucleotides in DNA has to be transcribed to
the sequence of aminoacids in polypeptide chain. It undergoes in two steps, tracription fo the
DNA sequence to the single stranded RNA (messenger, mRNA) and translation of the
nucleotide sequence to the correct sequence of aminoacids to produce protein for which a
given gene is responsible.
Transcription (DNA → RNA)
RNA polymerase - key enzyme, synthetizes all types of RNA, catalyzes all steps of the
process: recognizes the initial site for synthesis, unwinds the double helix of DNA, catalyzes
the addition of nucleotides, recognizes the termination site. Has no proof reading ability.
Postrancriptional processing
Prokaryotic mRNA- without processing
Eukaryotic mRNA
Splicing – excision of introns (intervening seguences)
Poly A tail on the 3’ end protection against rapid destruction by poly A binding proteins
Capping of the pre-mRNA involves the addition of 7-methylguanosine (m7G) to the 5' end.
The cap protects the 5' end of the primary RNA transcript from attack by ribonucleases that
have specificity to the 3'-5' phosphodiester bonds.
Translation (RNA → protein)
Order of aminoacids in polypetide chain is given by order of nucleotides in DNA, resp.
mRNA
Genetic code
4 nucleotide bases has to code for 20 amino acids - 43 = 64 combinations – triplets
Proteosynthesis
Occurs on ribosomes (small and big subunit )
Iniation factors
tRNA activation reaction → aminoacyl tRNA
Mechanism of polypeptide chain synthesis
Termination
Postranslation modification of proteins
Regulation of gene expression
Constitutive genes, inducible genes,
lac Operon
Brief overview of gene technologies
Recombinant technology
Lecture 8
BASIC CONCEPT OF METABOLISM AND ENERGY
CONVERSION
Metabolic Pathways
Catabolism: degradation of molecules to provide energy
Anabolism: reactions using energy to synthesize new molecules for growth etc.
Metabolic pathway is a series of enzyme-catalyzed reactions, initiated by a flux-generating
step and ending with either the loss of products to the environment, to a stored product (a
metabolic 'sink') or in a reaction that precedes another flux-generating step (that is, the
beginning of the next pathway)." Where a flux generating step is a non-equilibrium reaction
that generates the flux going through the pathway and to whose rate all other reactions of the
pathway conform. Note that by this definition some pathways may be inter-organ while others
may take place in single compartment. We will explore this definition/concept as we look at
metabolism.
Characteristics of pathways:
•
•
•
•
•
Irreversible
First committed step (flux generating step)
Regulated
Localized in eukaryotes
Catabolic and anabolic pathways are generally distinguished by coenzymes and/or
compartmentalization.
The flux through a metabolic pathway is invariably controlled or regulated, most commonly
by Feedback Inhibition, but also through Feed-forward activation. Regulation is one of the
things that makes biochemistry "biological" and it will be a focus in our study.
The stages of catabolism
For convenience we can breakdown catabolism into four hierarchical levels:
•
•
•
•
Stage I: Hydrolysis of polymers to monomeric units (fat
fatty acids and glycerol,
protein
amino acids, etc.)
Stage II: breakdown of products of Stage I to pyruvate, acetyl CoA, and/or
intermediates of the Kreb's Cycle
Stage III: Breakdown of Acetyl CoA by the Kreb's Cycle into carbon dioxide and
water with the production of reducing equivalents (NADH etc.)
Stage IV: Oxidation of the reducing equivalents by oxygen with the production of
ATP via the Electron Transport System.
Reactions in Metabolism
Organic Reaction Mechanisms: We can categorize all common biological reactions into four
groups:
•
•
•
•
Group-transfer reactions (transfer of an electrophile [acyl {RCOX}, phosphoryl
{OPO3X2-}, and glycosyl groups] between nucleophiles [alcohols, amines, thiols,
etc.])
Redox Reactions
Eliminations {eliminate H2O, NH3, ROH or RNH2}, Isomerizations, Rearrangements
C-C bond formation or breakage (condensation and cleavage reactions).
High Energy Compounds
Look at ATP:
Each of the phosphoric acid anhydride bonds is unstable. That is hydrolyzing either will
release a lot of energy.
So why ATP? First, we want a compound with intermediate hydrolysis energy so it can pick
up energy from some reactions and deliver to others. Second we want a kinetically stable
molecule which is thermodynamically unstable. Thus acetic acid anhydride would not
work: it is thermodynamically unstable to hydrolysis, but it is also kinetically unstable, with
the carbonyl carbons wide open to water attack. Phosphoric acid anhydride is equally
unstable, but is is sterically protected from water attack - in order to react quickly we need a
catalyst - perfect.
ATP is sometimes referred to as a "Hi Energy" compound. High energy in this case does not
refer to total energy in compound, rather just to energy of hydrolysis. Thus ATP is unstable to
hydrolysis, or has a large negative G for hydrolysis. For biochemistry High Energy is
defined in terms of ATP: if a compound's free energy for hydrolysis is equal to or greater than
ATP's then it is "High Energy," if its free energy of hydrolysis is less than ATP's then it is not
a "hi energy" compound. Note that ATP has two hi energy anhydride bonds.
Thermodynamics in Metabolism
Remember, the cool thing about thermo is that it is pathway independent - we can tell how
much energy is available in an M&M by burning it in pure oxygen in a stainless steel
container and tell you how far you can run on that M&M!
The tragedy of thermo, the other side of the coin, is that it tells nothing about the details thermo gives us no idea about how the energy is used, or what steps are involved in its loss.
Remember also that for chemists and biologists the thermodynamic term generally of most
interest is the Free Energy for a reaction, that is the energy available to do work.
•
•
•
The free energy is defined as: G = Gproducts- Greactants = H - T S.
When the free energy is negative we say the reaction is spontaneous, which simply
means the reactants are favored in the reaction equation as written.
Note when a reaction is at equilibrium then the G is zero.
Since free energy depends on conditions, chemists tabulate free energies under Standard
Conditions, ( G°): 298 K, 1 atm., with all concentrations at 1 M.
For biological systems we define a slightly different standard free energy with [H+]= 10-7 M
(pH=7), G° '.
For non-standard conditions we can find the free energy of a reaction using:
G = G° ' + RT lnQ.
For the special case of equilibrium, the free energy is zero, so
G° ' = -RT lnK',
Reaction must be exergonic ∆G < 0
Endergonic reaction can not run ∆G > 0 and must be realised by different way
Solution:
Lecture 9
ELECTROTRANSPORT SYSTÉM, CITRIC ACID
CYCLE
Electrotransport system
NADH and FADH2 carry protons (H+) and electrons (e-) to the electron transport chain
located in the membrane. The energy from the transfer of electrons along the chain transports
protons across the membrane and creates an electrochemical gradient. As the accumulating
protons follow the electrochemical gradient back across the membrane through an ATP
synthase complex, the movement of the protons provides energy for synthesizing ATP from
ADP and phosphate. At the end of the electron transport system, two protons, two electrons,
and half of an oxygen molecule combine to form water. Since oxygen is the final electron
acceptor, the process is called aerobic respiration.
ATP Production during Aerobic Respiration by Oxidative Phosphorylation
involving an Electron Transport System
Citric acid cycle
The citric acid cycle , also known as the tricarboxylic acid cycle (TCA cycle) and the Krebs
cycle is a series of enzyme-catalysed chemical reactions of central importance in all living
cells that use oxygen as part of cellular respiration.
TCA cycle
In eukaryotes, the citric acid cycle occurs in the matrix of the mitochondrion. In aerobic
organisms, the citric acid cycle is part of a metabolic pathway involved in conversion of
carbohydrates, fats and proteins into carbon dioxide and water to generace a form of usable
energy. Other relevant reactions in the pathway include those in glycolysis and pyruvate
oxidation before the citric acid cycle, and oxidative phosphorylation after it. In addition, it
provides precursors for many compounds including some amino acids and is therefore
functional even in cells performing fermentation.
Lecture 10
METABOLISM OF CARBOHYDRATES
Wider metabolic context of glycolysis
The Energy Derived from Glucose Oxidation
Aerobic glycolysis of glucose to pyruvate, requires two equivalents of ATP to activate the
process, with the subsequent production of four equivalents of ATP and two equivalents of
NADH. Thus, conversion of one mole of glucose to two moles of pyruvate is accompanied by
the net production of two moles each of ATP and NADH.
Glucose + 2 ADP + 2 NAD+ + 2 Pi ——> 2 Pyruvate + 2 ATP + 2 NADH + 2 H+
The NADH generated during glycolysis is used to fuel mitochondrial ATP synthesis via
oxidative phosphorylation, producing either two or three equivalents of ATP.
Glycolysis
Regulation of Glycolysis
The reactions catalyzed by hexokinase, PFK-1 and PK all proceed with a relatively large free
energy decrease. These non-equilibrium reactions of glycolysis would be ideal candidates for
regulation of the flux through glycolysis. Indeed, in vitro studies have shown all three
enzymes to be allosterically controlled.
Regulation of hexokinase, however, is not the major control point in glycolysis. This is due to
the fact that large amounts of G6P are derived from the breakdown of glycogen (the
predominant mechanism of carbohydrate entry into glycolysis in skeletal muscle) and,
therefore, the hexokinase reaction is not necessary. Regulation of PK is important for
reversing glycolysis when ATP is high in order to activate gluconeogenesis. As such this
enzyme catalyzed reaction is not a major control point in glycolysis. The rate limiting step in
glycolysis is the reaction catalyzed by PFK-1.
Alternative pathways
Gluconeogenesis
Gluconeogenesis is the biosynthesis of new glucose, (i.e. not glucose from glycogen). The
production of glucose from other metabolites is necessary for use as a fuel source. The
primary carbon skeletons used for gluconeogenesis are derived from pyruvate, lactate,
glycerol, and the amino acids alanine and glutamine. The liver is the major site of
gluconeogenesis.
The relevant reactions of gluconeogenesis are depicted. The enzymes of the 3 bypass steps are
indicated in green along with phosphoglycerate kinase. This latter enzyme is included since
when functioning in the gluconeogenic direction the reaction consumes energy.
Gluconeogenesis from 2 moles of pyruvate to 2 moles of 1,3-bisphosphoglycerate consumes 6
moles of ATP.
Lecture 11
PHOTOSYNTHESIS
Photosynthesis is an important biochemical process in which plants, algae, protistans, and
some bacteria convert the energy of sunlight to chemical energy and store it in the bonds of
sugar, glucose.
Ultimately, nearly all living things depend on energy produced from photosynthesis for their
nourishment, making it vital to life on Earth. It is also responsible for producing the oxygen
that makes up a large portion of the Earth's atmosphere. Organisms that produce energy
through photosynthesis are called photoautotrophs. Plants are the most visible representatives
of photoautotrophs, but it should be emphasized that bacteria and algae as well contribute to
the conversion of free energy into usable energy.
Light-dependent reactions
The reaction of all light-dependent reactions in oxygenic photosynthesis is:
12H2O + 12NADP+ + 18ADP + 18Pi → 6O2 + 12NADPH + 18ATP
The light-dependent reactions, or light reactions, are the first stage of photosynthesis. In
this process light energy is converted into chemical energy, in the form of the energy-carriers
ATP and NADPH. In plants, the light-dependent reactions occur in the thylakoid
membranes of the chloroplasts and use light energy to synthesize ATP and NADPH.
The photons are captured in the antenna complexes of photosystem I and II by chlorophyll
and accessory pigments (see diagram at right). When a chlorophyll a molecule at a
photosystem's reaction center absorbs energy, an electron is excited and transferred to an
electron-acceptor molecule through a process called Photoinduced charge separation. These
electrons are shuttled through an electron transport chain that initially functions to generate a
chemiosmotic potential across the membrane, the so called Z-scheme shown in the diagram.
An ATP synthase enzyme uses the chemiosmotic potential to make ATP during
photophosphorylation while NADPH is a product of the terminal redox reaction in the Zscheme.
Water photolysis
The NADPH is the main reducing agent in chloroplasts, providing a source of energetic
electrons to other reactions. Its production leaves chlorophyll with a deficit of electrons
(oxidized), which must be obtained from some other reducing agent. The excited electrons
lost from chlorophyll in photosystem I are replaced from the electron transport chain by
plastocyanin. However, since photosystem II includes the first steps of the Z-scheme, an
external source of electrons is required to reduce its oxidized chlorophyll a molecules. This
role is played by water during a reaction known as photolysis and results in water being split
to give electrons, oxygen and hydrogen ions. Photosystem II is the only known biological
enzyme that carries out this oxidation of water. Initially, the hydrogen ions from photolysis
contribute to the chemiosmotic potential but eventually they combine with the hydrogen
carrier molecule NADP+ to form NADPH. Oxygen is a waste product of light-independent
reactions, but the majority of organisms on Earth use oxygen for cellular respiration,
including photosynthetic organisms.
Calvin cycle
The Calvin cycle or Calvin-Benson-Bassham cycle is a series of biochemical reactions that
take place in the stroma of chloroplasts in photosynthetic organisms. It was discovered by
Melvin Calvin, James Bassham and Andrew Benson at the University of California,
Berkeley.[1] It is one of the light-independent (dark) reactions, used for carbon fixation.
Steps of Calvin cycle
1. The enzyme RuBisCO catalyses the carboxylation of Ribulose-1,5-bisphosphate a 5carbon compound, by carbon dioxide (a total of 6 carbons) in a two-step reaction. The
initial product of the reaction is a six-carbon intermediate so unstable that it
immediately splits in half, forming two molecules of glycerate -3-phosphate, a 3carbon compound. (also: 3-phosphoglycerate, 3-phosphoglyceric acid, 3PGA)
2. The enzyme phosphoglycerate kinase catalyses the phosphorylation of 3PGA by ATP
(which was produced in the light-dependent stage). 1,3 bisphosphoglycerate and ADP
are the products. (However, note that two PGAs are produced for every CO2 that
enters the cycle, so this step utilizes 2 ATP per CO2 fixed).
3. The enzyme G3P dehydrogenase catalyses the reduction of 1,3BPGA by NADPH
(which is another product of the light-dependent stage). Glyceraldehyde 3-phosphate
(also G3P, GP, TP, PGAL) is produced, and the NADPH itself was oxidized and
becomes NADP+. Again, two NADPH are utilized per CO2 fixed.
The enzymes in the Calvin cycle are functionally equivalent to many enzymes used in other
metabolic pathways such as gluconeogenesis and the pentose phosphate pathway, but they are
to be found in the chloroplast stroma instead of the cell cytoplasm, separating the reactions.
They are activated in the light (which is why the name "dark reaction" is misleading), and also
by products of the light-dependent reaction. These regulatory functions prevent the Calvin
cycle from being respired to carbon dioxide. Energy (in the form of ATP) would be wasted in
carrying out these reactions that have no net productivity.
The sum of reactions in the Calvin cycle is the following:
3 CO2 + 6 NADPH + 5 H2O + 9 ATP → Glyceraldehyde 3-Phosphate(G3P) + 2 H+ +
6 NADP+ + 9 ADP + 8 Pi
It should be noted that hexose (six-carbon) sugars are not a product of the Calvin cycle.
Although many texts list a product of photosynthesis as C6H12O6, this is mainly a convenience
to counter the equation of respiration, where six-carbon sugars are oxidized in mitochondria.
The carbohydrate products of the Calvin Cycle are three-carbon sugar phosphate molecules,
or "triose phosphates," to be specific, glyceraldehyde-3-phosphate (G3P).
Comparison photosynthesis and respiration
Lecture 12
METABOLISM OF LIPIDS
Lipids are a broad group of naturally occurring compounds either non-polar or amphipathic
defined by low solubility in water.
Triacyl glycerols (fats)
Glycerol esterified with three fatty acids, serve as metabolic energy store of important fuel
molecules fatty acids:
Common saturated fatty acids in organisms
Symbol
Common
formula
Melting
name
point
(°C)
12:0
lauric
CH3(CH2)10COOH
44.2
14:0
myristic
CH3(CH2)12COOH
52
16:0
palmitic
CH3(CH2)14COOH
63.1
18:0
stearic
CH3(CH2)16COOH
69.6
20:0
arachidonic CH3(CH2)18COOH
75.4
Common unsaturated fatty acids in organisms
Symbol
fomula
16:1∆9
18:1 ∆9
18:2 ∆9,12
Common
name
Palmitoleic
oleic
linoleic
CH3(CH2)5CH=CH-(CH2)7COOH
CH3(CH2)7CH=CH-(CH2)7COOH
CH3(CH2)4(CH=CHCH2)2(CH2)6COOH
Melting
point (°C)
-0.5
13.4
-9
18:3 ∆9,12,15
linolenic
CH3CH2(CH=CHCH2)3(CH2)6COOH
-17
20:4
∆5,8,11,14
arachidonic
CH3(CH2)4(CH=CHCH2)4(CH2)2COOH
-49
Waxes – esters of long-chain primary alcohols and long-chain fatty acid, this class of
molecules confers water-repellant character to animal skin, to the leaves of certain plants, and
to bird feathers.
Polar lipids
Phospholipids (glycerophospholipids) and sphingolipids constituents of biological
membranes due to their amphiphilic character. Non polar region of the phospholipid
molecule is formed by two acyl chains of fatty acids, polar part of the molecule by the
phosphate (phosphatidic acid) esterified with various alcohols (choline, ethanolamine, serine,
inositol)
Sphingolipids are based on aminoalcohol sphingosine (instead of glycerol in
glycerophospholipds) modified with one acyl chain and polar part is formed by hydrogen (
ceramide) phosphocholine (sphingomyeline) or saccharide residue (glucosylcerebroside or
ganglioside).
Polar lipids in contact with water spontaneously
form the lipid vesicles, micelles or bilayers, basic
process of biological membrane formation.
Isoprenoids – derived from isoprene units (2methy-1,3-butadiene)
Biologically active compounds and could be
divided to two groups sterols and terpenes
Steroids are characterized by fused ring system.
Cholesterol is the best known steroid found in
plasma lipoproteins and serving as membrane lipid
or a
precursor of steroid hormones, (testosterone,
estradiol, cortisol) or vitamin D.
Terpenes are a class of lipids formed from combinations of two or more molecules isoprene,
linear and cyclic compounds – carotene, retinol, squalene, lycopene, gibberelic acid etc.
Eicosanoids – derivatives of arachidonic acid of three subclasses: prostaglandins,
thromboxanes, leukotrienes and lipoxins– they posses hormonlike signaling function
(regulation of blood pressure and blood clotting, generarion of inflammation, fever and pain)
Lipid metabolism
Metabolism of triacylglycerol
Diatary or synthetized in liver and stored in adipocytes or myocytes.
Digestion of dietary triacylglycrols starts in small intestine, emulsified with bile salts, cleaved
by pancreatic lipase to monoacylglycerol and free fatty acids, transported to intestinal
mucosa, whwre they are resynthetized to triacylglycerols and transported in the form of
lipoprotein particles (chylomicrons) by blood stream to the stores.
Katabolism of triacylglycerols
Cleaved by lipase to glycerol and free fatty acids
Glycerol is further metabolised to the intermediates of glucose metabolism
Fatty acids are activated in cytoplasm by the reaction with CoA with concomitant cleavage of
ATP (fatty acid –CoA ligase)
Activated fatty acid is transferred to
carnitine and acylcarnitin is transported by
palmitoyl transferase I and II located in the
inner mitochondrial membrane to the
mitochondrial matrix where acyl carnitine
reacylates the mitochondrial CoA.
Acyl CoA then undergoes the degradation
through the β-oxidation, a spiral pathway.
In each turn the acyl chain undergoes a
four step sequence of dehydrogenation,
hydration, second dehydrogenation and
thiolytic cleavage of C-C bond forming
the acetyl CoA and acyl CoA of the fatty
acid shorter by two carbon atoms
Reduced cofactors NADH + H+ and
FADH2 are reoxidized in electron
transport chain coupled with oxidative
phosphorylation (formation of ATP) in the
inner mitochondrial membrane
Acetyl CoA could be further metabolized
in citric acid cycle.
The overall energy yield (number of ATP
molecules) of complete oxidation of the
n ⎛n ⎞
fatty acid with n carbon atoms is then: 12 + 5⎜ − 1⎟ − 2
2 ⎝2 ⎠
β-oxidation of unsaturated fatty acids
β-oxidation of fatty acids with odd number of carbon atoms
Metabolism of keton bodies
Biosynthesis of fatty acids
Fatty acid synthesis from acetyl
CoA occurs in cytoplasm. Acetyl
CoA is carboxylated to malonyl
CoA (-OOCCH2COSCoA) by
acetyl CoA carboxylase. Fatty
acid synthesis is catalyzed by
fatty acid synthase complex (in
animals) and begins with acetyl
CoA and malonyl CoA which are
bound to acyl carrier protein
(ACP), part of the enzyme
complex. These two molecules
undergous the condensation
reactin
with
concomitant
decarboxylation followed by
reduction, dehydrogenation and
another
reduction.
These
reactions can be considered as
the reverse of β-oxidation. The
reduction agent is NADPH + H+.
Product of the first cycle is the
four carbon butyryl CoA which
enters the first reaction and
condensate with another malonyl
CoA. During each run the chain
elongates by two carbons. In
animal cells the process stops at 16 carbon palmitic acid. Elongation of palmitate as well as
introduction of double bonds must be carried out by other enzyme systems.
Biosynthesis of triacylglycerol – precursor is glycerol 3-phosphate which is acylated by acyl
CoA to monoacylglycerol 3-phosphate and subsequently to diacylglycerol 3-phosphate, this
is dephosphorylated to diacylglycerol and acylated to triacylglycerol.
Glycerophospholipid biosynthesis – pathway for the synthesis vary with type of the
organism. Starts with daicylglycerol or diacylglycerol 3-phosphate. CDP serves as carrier of
polar head (CDP-choline, CDP-ethanolamine.
Isoprenoids (steroids) – mevalonate pathway
Lecture 13
METABOLISM OF NITROUS COMPOUNDS
Nitrogen in the nature
•
•
•
•
•
N2 abundant, fixation by bacteria = reduction to NH3 (NH4+)
nitrification- soil (NH4+) oxidized to nitrite and nitrate
plants and many bacteria convert nitrate and nitrite to (NH4+) and amino acids, etc.
animals get amino acids from plants
denitrification- conversion of nitrates to N2
Metabolism of nitrogen
Urea cycle
The urea cycle takes place in the liver. The steps in the urea cycle occur in two places in the
cells of the liver: the mitochondria and the cytosol within the cytoplasm.
The first three steps in the urea cycle (N-acetylglutamine Synthase, Carbamyl Phosphate
Synthetase and Ornithine Transcarbamylase) occur in the mitochondria of the cell. The
mitochondria contain the metabolic pathways involved in the metabolism of the
carbohydrates, lipids and amino acids. The special pathways involving heme and urea
synthesis are also located in the mitochondria. It generates most of the cell's ATP that
provides the energy for the metabolic pathways to occur.
The last three steps of the urea cycle (Argininosuccinate Synthetase, Argininosuccinate
Synthase Lyase, and Arginase) occur in the cytosol. Cytosol is the aqueous solution that
makes up the cytoplasm. It contains thousands of enzymes involved in intermediate
metabolism and ribosomes making proteins.
Central role of glutamate:
Four of the amino acids: glutamate, aspartate, alanine and glutamine are present in
mammalian cells at much higher concentrations than the other 16. All four have major
metabolic functions in addition to their roles in proteins, but glutamate occupies the prime
position.
Glutamate and Glutamine
Glutamine Synthetase
Ubiquitous- all organisms
1. glutamate + ATP --> γ-glutamyl phosphate + ADP
2. γ-glutamyl phosphate + NH4+ --> glutamine + Pi
Glutamate Synthetase
Plants and bacteria; animals use transamination to α-KG during AA catabolism
α-KG + glutamine + NADPH + H+ --> 2 glutamate + NADP+
sum; α-KG + NH4+ + NADPH + ATP --> l-glutamate + NADP+ + ADP + Pi
Glutamate Dehydrogenase
mitochondria, 1 mM Km for NH4+ makes reverse likely
α-KG + NH4+ + NADPH --> L-glutamate + NADP+ + H2O
Glutamate and aspartate function as excitatory neurotransmitters in the central nervous
system, and glutamate is partly responsible for the flavour of food. (It is the mono sodium
glutamate listed on processed food labels.) However, glutamate also occupies a special
position in amino acid breakdown, and most of the nitrogen from dietary protein is ultimately
excreted from the body via the glutamate pool.
Glutamate is special because it is chemically related to 2-oxoglutarate ( = a-ketoglutarate)
which is a key intermediate in the Krebs cycle. Glutamate can be reversibly converted into 2oxoglutarate by transaminases or by glutamate dehydrogenase. In addition, glutamate can be
reversibly converted into glutamine, an important nitrogen donor, and the most common free
amino acid in human blood plasma.
Transamination reactions:
Most common amino acids can be converted into the corresponding keto acid by
transamination. This reaction swops the amino group from one amino acid to a different keto
acid, thereby generating a new pairing of amino acid and keto acid. There is no overall loss or
gain of nitrogen from the system - it is simply a question of "robbing Peter to pay Paul".
Transamination reactions are readily reversible, and the equilibrium constant is close to 1.
One of the two substrate pairs is usually glutamate and its corresponding keto acid oxoglutarate. All transaminases require pyridoxal phosphate or pyridoxamine phosphate (both
derived from vitamin b6) as an essential cofactor.
The reaction mechanism is shown on the next page. The substrates bind to the enzyme active
centre one at a time, and the function of the pyridoxal phosphate is to act as a temporary store
of amino groups until the next substrate comes along. In the process the pyridoxal phosphate
is converted into pyridoxamine phosphate, and then back again. Enzymologists call this a
"ping pong" mechanism, and it leads to a characteristic pattern of parallel lines in a double
reciprocal plot of 1/V versus 1/S1 at various S2 concentrations.
The condensation between the alpha amino group and the aromatic aldehyde to form a "Schiff
base" makes the alpha carbon atom chemically reactive, so the isomerisation of the Schiff
base takes place very easily. In practice the pyridoxal form of the coenzyme condenses with
the epsilon amino group of a lysine residue in the enzyme protein when no amino acid is
bound, and the free aldehyde form of the coenzyme has only a transitory existence. Many of
the enyzmes that metabolise amino acids require pyridoxal phosphate as a cofactor.
Unexpectedly, this compound also serves in a completely different manner in the active centre
of glycogen phosphorylase.
Glycogenic and ketogenic amino acids
The carbon skeletons from the majority of amino acids are degraded to Krebs cycle
intermediates after removal of the amino group by transamination. This means that they can
give rise to blood glucose via the gluconeogenic pathway (Dr Bonnett’s lectures). They are
termed 'glycogenic' amino acids, because it was observed many years ago that they made
diabetic glycosuria worse. In contrast to this 'ketogenic' amino acids exacerbated diabetic
ketoacidosis, and these amino acids are degraded to compounds such as acetoacetate and
acetyl-CoA. 'Mixed' amino acids are degraded to both Krebs cycle acids and to acetyl-CoA.
The situation is summarised in the following table:
Phenylketonuria:
Phenylalanine is normally metabolised by conversion to tyrosine. The enzyme responsible for
this conversion is phenylalanine hydroxylase, a mixed function oxygenase with a
tetrahydrobiopterin cofactor:
Half of the oxygen molecule re-appears in the tyrosine -OH group and the other half is
reduced to water. The "dihydrobiopterin" in the above reaction is an isomer of the folic acid
compounds involved in one-carbon metabolism. It is recycled back to tetrahydrobiopterin
using NADH:
"dihydrobiopterin" + NADH = tetrahydrobiopterin + NAD
Approximately one person in 45 American whites is a carrier for a defective phenylalanine
hydroxylase gene, or (less frequently) the dihydrobiopterin cofactor. These mutations are
particularly common in people of Celtic origin, but are less frequent in Eastern Europe.
Unless treated they are seriously mentally defective and excrete large quantities of
phenylpyruvate in the urine. This compound gives the disease its name, and is formed by the
transamination of phenylalanine, a reaction that is normally insignificant. These patients are
often tyrosine deficient, and have abnormally light skin pigmentation, because they have
insufficient tyrosine to synthesise melanin in normal amounts.
When this condition was first recognised in the 1930's, a significant proportion of all long
term patients in mental institutions proved to be undiagnosed phenylketonuriacs. Nowadays
the condition can be readily diagnosed by a heel-prick blood test performed on all new-born
babies.
Treatment consists of a very low phenylalanine diet, supplemented with extra tyrosine that the
patients cannot synthesise from phenylalanine. This diet must be instituted at birth, but can be
discontinued after a few years when brain maturation is completed. For obvious reasons it
must be restarted during pregnancy. The artificial sweetner aspartame is a phenylalanine
derivative, and this fact is declared on soft drinks cans to assist those following the special
diet.
Lecture 14
REGULATION OF METABOLIC PATHWAYS
Regulation of methabolic pathways will be shown directly on examples from former lectures.
Discussion will be focused on different levels of regulation
-
allosteric effect
regulation by phosphorylation
Example of regulation after being scared by angry bear