The investigation of active site structure: The identification of binding sites and catalytic

sites.
–trapping the E-S complex, use of substrate analogs, enzyme modification by treatment with
proteolytic enzymes, photo – oxidation and chemical modification of amino acid side chains
(cys, met, his, ser, asp, glu, lys, and tyr). Affinity labeling studies (chymotrypsin
triosephosphate isomerase) an and super reactive amino acid chains (chymotrypsin and
glutamatedehydrogenase). Site directed mutagenesis. The 3-D structural features of active
sites of chymotrypsin trypsin, elastase and triose phosphate isomerase.
6 hrs

Mention the characteristic features of active site of an enzyme.

The active site of an enzyme is the region that binds the substrates. It also
contains the residues that directly participate in the making and breaking of
bonds. In essence, the interaction of the enzyme and substrate at the active site
promotes the formation of the transition state.
The active site is the region of the enzyme that most directly lowers the energy
of Activation of the reaction, which results in the rate enhancement
of enzyme action.
1. The active site occupies a relatively small portion of the enzyme molecule.
2. The active site is neither a point nor a line or even a plane but is a 3-
dimensional entity. It is made up of groups that come from different parts of the
linear amino acid sequence. For example, as already stated, lysozyme has 6
subsites in the active site. The amino acid residues located at the active site are
35, 52, 59, 62, 63 and 107.
3. Usually the arrangement of atoms in the active site is well defined, resulting
in a marked specificity of the enzymes. The active site changes its configuration
in order to bind a substance which is only slightly different in structure from its
own substrate.
4. The active site binds the substrate molecule by relatively weak forces.
5. The active sites in the enzyme molecules are grooves or crevices from which
water is largely excluded. It contains amino acids such as aspartic acid, glutamic
acid, lysine serine etc. The side chain groups like -COOH, NH2, CH2OH etc.,
serve as catalytic groups in the active site.
6. The crevice creates a micro-environment in which certain polar residues
acquire special properties which are essential for catalysis

Page 1 of 32
Trapping of ES complex

 Principle: It involves methods to trap the ES complex and identify the

amino acids that are bound to the ES complex.
 EX: Oxidative decarboxylation of Iscitrate to ketoglutarate by Isocitrate
dehydrogenase.
 By trapping the ES complex, the amide group of nicotinamide, NADP
is found to be interacting with the carboxyl group of isocitrate and the
carboxyl group of Glu-336, amino group of Lys-100, and the side chain
of Asn-115 .

The enzyme proceeds through a multi step reaction pathway, ultimately

converting isocitrate and NAD(P) to Y ketoglutarate (KG). CO2, and NAD(P)H.

Use of substrate analogs: Substrate analogs are molecules which can be

inhibitors and can involve in the inactivation of enzymes by extremes of pH,
temperature or chemical reagents.
Most frequently, however, it causes the covalent modification of the enzyme in
a manner that destroys activity.

Ex: The inhibition of enzymes involved in bacterial cell wall synthesis is

exploited through the use of penicillin as an antibiotic that binds to bacterial
enzymes involved in cell wall synthesis causing inactivation.

Other examples are listed in the table:

Page 2 of 32
Active site investigation by the action of proteases
The precursors should be converted to the active forms before they can catalyze
a chemical reaction. This type of activation usually involves cleavage of the
peptide bond .
:
Ex: Chymotrypsin is synthesized by the exocrine cells of the pancreas in its
precursor form, chymotrypsinogen. Hydrolysis by trypsin converts
chymotrypsinogen into the active form, chymotrypsin.

Proteolytic activation of chymotrypsinogen

Chemical modification of aminoacid side chain

 A number of chemicals are known to selectively modify specific amino
acid side chains within proteins .
 Some of these that are commonly used to study enzyme active sites.

Page 3 of 32
 These compounds covalently modify the accessible amino acids in a
general way, so that treatment of an enzyme with such reagents will lead
to modification of both catalytically critical residues and nonessential
residues.

Page 4 of 32
 Affinity Labelling: Principle : modifying the aminoacid side chain by
labelling it and incorporating into substrate specific functional sites on
the enzyme molecule. The concept of affinity labelling of an enzyme is
used to study enzyme active site and mechanism.

 This approach can help to identify key aminoacid residues of an enzyme

 Certain reactive molecules will react selectively with specific amino
acid side chains, and these radiolabelled and incorporated into substrate
molecules.

 Ex: Affinity labelling studies using bromo hydroxyacetone phosphate

modified a glutamate residue at the active site of Triose phosphate
isomerise (Ala-Tyr-Glu-Pro-Val- Trp) and this residue was identified as
Glu165.

 Glutamate dehydrogenase (MW 330,000) is a complex allosteric

enzyme and is present only in the mitochondrial matrix. The enzyme
molecule consists of 6 identical subunits.
 Glutamate dehydrogenase from Escherichia coli (MW = 50,000) is a
hexamer of 6 identical subunits. In eukaryotic cells, L-glutamate
dehydrogenase is located in the mitochondrial matrix.
 It is influenced by the positive modulator ADP and by the negative
modulator GTP for making α-ketoglutarate available for the citric acid
cycle and releasing NH4 + for excretion.

Site directed mutagenesis:

 Site-directed mutagenesis is the technique for altering the amino acid
residue in an enzyme protein by mutation to study the activity of the
mutated enzyme site-directed mutagenesis in which one amino acid
residue is substituted for another have allowed to pinpoint the chemical
groups that participate in enzyme catalysis..
Page 5 of 32
  The catalytic importance of the active site serine and histidine residues
has been demonstrated by site-directed mutagenesis studies, in which
replacement of either the serine or the histidine or both reduced the rate
of reaction by the enzyme by as much as 10 fold.

 For example, in an enzyme protein in which a His 323, Asn was

introduced by means of site-directed mutagenesis. This means that in the
natural, or wild-type protein, a histidine residue occupies position 323,
but through mutagenesis this residue has been replaced by an asparagine
to create a mutant (altered) protein. Which showed a reduced activity.

 Amongst the residues forming the active sites are Tyr98, Ser136 Trp153,
His183, Glu190, Leu194, Tyr195, Gly225 and Leu226 with site directed
mutagenesis suggesting that Tyr98, His183, and Leu194 are particularly
important in forming critical hydrogen bonds with the substrate

3D Stucture of Chymotrypsin, Trypsin ,Elastase and Trise phosphate isomerase

 Chymotrypsin is a good example of the use of covalent modification as a

catalytic strategy.
 The enzyme employs a powerful nucleophile to attack the unreactive carbonyl
group of the substrate.
 This nucleophile becomes covalently attached to the substrate briefly in the
course of catalysis.
 The active site of chymotrypsin, marked by serine 195, lies in a cleft on the
surface of the enzyme.
 The structural analysis revealed the chemical basis of the special reactivity of
serine 195 . The -NH group of this imidazole ring is, in turn, hydrogen bonded
to the carboxylate group of aspartate 102. This constellation of residues is
referred to as the catalytic triad.

Page 6 of 32
  The catalytic triad, shown on the left, converts serine 195 into a potent
nucleophile, as illustrated on the right.

 The side chain of serine 195 is hydrogen bonded to the imidazole ring of
histidine 57.
 The arrangement of residues lead to the high reactivity of serine 195. The
histidine residue serves to position the serine side chain and to polarize its
hydroxyl group.

 In doing so, the residue acts as a general base catalyst, a hydrogen ion acceptor,
because the polarized hydroxyl group of the serine residue is poised for
deprotonation.
 The withdrawal of the proton from the hydroxyl group generates an alkoxide
ion, which is a much more powerful nucleophile than an alcohol is.

 The aspartate residue helps orient the histidine residue and make it a better
proton acceptor through electrostatic effects.

Role of the catalytic triad in chymotrypsin

 Catalytic Triad. Proteolytic enzymes containing a highly reactive serine
residue are known as serine proteases. These enzymes are readily
identifiable by their rapid inactivation by DIPF.
 Chymotrypsin, trypsin and thrombin are noteworthy examples of this
clan.
 Chymotrypsin contains 28 seryl residues but only one of them (Ser195) is
a strong nucleophile.
 This is due to a specific spatial relationship between three amino acid
residues (His57, Asp102 and Ser195) which constitute a catalytic triad
and is based on hydrogen bonding .

Page 7 of 32
  The hydrogen bonding, that occurs between the buried Asp102and His57
and between His57 and Ser195, establishes an equilibrium that allows for
the loss of the proton of the OH group of Ser195 (at the catalytic site) to
His57. This loss makes the oxygen atom of Ser195 residue a strong
nucleophile, i.e., makes serine195 an active serine.
 The proton gained by His57 converts the side chain of that residue into a
positive imidazolium ion that forms an ion pair with the negative
carboxylate ion of Asp102. Thus, loss of a proton by Ser195 and
formation of an ion pair by His57 and Asp102 explain the mechanism
behind the functioning of catalytic triad.

The active site Pockets of Chymotrypsin, Trypsin, and Elastase. Certain residues
play key roles in determining the specificity of these enzymes. The side chains of
these residues, as well as those of the active-site serine residues,

Page 8 of 32
The Oxyanion Hole is the structure stabilizes the tetrahedral intermediate of the
chymotrypsin reaction.
Hydrogen bonds link peptide NH groups and the negatively charged oxygen.

The side chains of the catalytic triad residues, including serine 195, has two
intrastrand and interstrand disulfide bonds

 The strategy used by the cysteine proteases is most similar to that used by the
chymotrypsin family. In these enzymes, a cysteine residue, activated by a
histidine residue, plays the role of the nucleophile that attacks the peptide bond
, in a manner quite analogous to that of the serine residue in serine proteases.

 An ideal example of these proteins is papain, an enzyme purified from the

fruit of the papaya.
 Mammalian proteases homologous to papain have been discovered, most
notably the cathepsins, proteins having a role in the immune and other systems.
 The cysteine based active site arose independently at least twice in the course
of evolution
 ; the caspases, enzymes that play a major role in apoptosis have active sites
similar to that of papain, but their overall structures are unrelated

Page 9 of 32
Outline the structural differences between Chymotrypsin, Trypsin and Elastase

The X-ray structures of the above three enzymes suggest the basis for their differing
substrate specificities :

1. In chymotrypsin, the bulky aromatic side chain of the preferred Phe, Trp, or Tyr
residue that contributes the carbonyl group of the scissile peptide fits snugly into a
slitlike hydrophobic pocket located near the catalytic groups.
2. In trypsin, the residue corresponding to chymotrypsin Ser 189, which lies at the
bottom of the binding pocket, is the anionic residue Asp.

The cationic side chains of trypsin’s preferred residues, Arg and Lys, can therefore
form ion pairs with this Asp residue. The rest of chymotrypsin’s specificity pocket is
preserved in trypsin so that it can accommodate the bulky side chains of Arg and
Lys .

3. Elastase is so named because it rapidly hydrolyzes the otherwise nearly

indigestible Ala, Gly, and Val–rich protein elastin (a major connective tissue
component). Elastase’s binding pocket is largely occluded by the side chains of Val
and Thr residues that replace the Gly residues lining the specificity pockets in both
chymotrypsin and trypsin.

Consequently elastase, whose substrate-binding site is better described as merely a

depression, specifically cleaves peptide bonds after small neutral residues,
particularly Ala.
In contrast, chymotrypsin and trypsin hydrolyze such peptide bonds extremely
slowly because these small substrates cannot be sufficiently immobilized on the
enzyme surface for efficient catalysis to occur.

Triose phosphate isomerise

 Triose phosphate isomerase has been widely studied and catalyses the
conversion between DHAP and G3P .
 The equilibrium for the reaction lies in favour of DHAP, but the rapid removal
of G3P by successive reactions in glycolysis drives the reaction towards the
formation of G3P.

 Triose phosphate isomerase merits further discussion as an example of enzyme

catalysis because of two interesting phenomena:
1) The use of the imidazolate form of histidine in catalysis and the evolution
of the enzyme towards a near perfect biological catalyst. Influencing both of

Page 10 of 32
 these phenomena is the structure of triose phosphate isomerase with
crystallographic structures

2) The interconversion of dihydroxyacetone phosphate (left) and glyceraldehyde-3-

phosphate catalysed by triose phosphate isomerise of the enzyme available in a free
state, bound with DHAP and plus 2-phosphoglycolate.

 Kinetic studies on the pH dependence of the catalytic process revealed two

inflection points in the profile corresponding to pH values of 6.0 and 9.0.
 When considered alongside the structure of triose phosphate isomerise these
values implicated the side chains of glutamate and histidine.

 Affinity labelling studies using bromohydroxyacetone phosphate modified a

glutamate residue within a hexapeptide (Ala-Tyr-Glu-Pro-Val-Trp) and this
residue was identified as Glu165.

 The active site lies near the top of the β barrel with Glu165 close to His 95 as
well as Lys12, Glu97 and Tyr208.
 Both Glu165 and His 95 are essential for catalytic activity and the enzyme
provides a cage or reaction vessel holding substrate and protecting

Page 11 of 32
 intermediates through a series of 11 residues immediately after Glu165
(residues 166–176) that form a loop and acts
 as a lid for the active site.

 Dihydroxyacetone phosphate binds to the active site with the carbonyl oxygen
forming a hydrogen bond with the neutral imidazole side chain of His95.

 The glutamate side chain is charged and facilitates abstraction of a proton from
the C1 carbon of dihydroxyacetone phosphate. In this state the His95 forms a
strong hydrogen bond with the carbonyl oxygen (C2) of the enediolate
intermediate that leads to protonation of this atom.

 Protonation of the oxygen atom forms an imidazolate side chain on His95 that
has a negative charge and is a strong base.

Proton abstraction from the hydroxyl group attached to the C1 carbon results in
the formation of a second enediolate intermediate with this unstable
intermediate converted to the final product by donation of a proton from
Glu165 to the C2 carbon.

Page 12 of 32
Syllabus

Enzyme catalysis: Chemical nature of enzyme catalysis and mechanism of -General acid-
base catalysis, electrostatic catalysis, covalent catalysis, intramolecular catalysis and enzyme
catalysis.
Mechanisms of action of the following enzymes-
Monomeric enzymes; serine proteases (chymotrypsin, trypsin, elastase),
sulphydryl enzymes (papain, lysozyme, ribonuclease,oligomeric enzymes lactate dehydrogenase )
and alcohol dehydrogenase
multi- enzyme complexes. (pyruvatedehdrogenase complex).
Metal– activated and metallo-enzymes (mechanism of action of pyruvate kinase, creatine kinase,
superoxide dismutase & carboxypeptidase – A) and multifunctional enzymes.
Artificial enzymes, Abyzmes- Catalytic antibodies.

The way enzymes work can be shown by looking at the energy changes during a
chemical reaction.
In a reaction where the product has a lower energy than the substrate, the substrate
naturally turns into product (i.e. the equilibrium lies in the direction of the product).

Before it can change into product, the substrate must overcome an "energy barrier"
called the activation energy. The larger the activation energy is, the slower the
reaction will be.
This is because only a few substrate molecules will have sufficient energy to
overcome the activation energy barrier. Imagine pushing boulders over a hump before
they can roll down hill, and you have the idea.

Most biological reactions have large activation energies, so they without enzymes
they happen far too slowly to be useful.

Enzymes reduce the activation energy of a reaction so that the kinetic energy of most
molecules exceeds the activation energy required and so they can react.

For example, for the catalase reaction (2H2O2 → 2H2O + O2) the activation energy
is 86 kJ mol -1 with no catalyst, 62 kJ mol -1 with an inorganic catalyst, and just 1 kJ
mol -1 with the enzyme catalase.

Page 13 of 32
Enzymes commonly employ one or more of the following strategies to catalyze
specific reactions:

In general acid-base catalysi

Acid/base Catalysis: Much of the instability of a typical transition state is due to the
build up of unfavourable charges within the molecule as bonds form and break.

 . Ribonuclease is a classic example of acid/base catalysis,

 Ribonuclease , a small globular protein, is an enzyme secreted by the

pancreas into the small intestine, where it catalyzes the hydrolysis of
certain bonds in ribonucleic acids.
 This enzyme-protein consists of a single polypeptide chain of 124 amino
acid residues with lysine at the N-terminal and valine at the C-terminal .
 The active site is believed to be on the edge of this depression and the residues
forming the active site are 6-8, 11, 12, 41, 42, 46-48 and 117-119.

 Ribonuclease catalyses the hydrolysis of RNA in a two-step process during

which a cyclic intermediate is formed .

 The uncatalysed reaction involves the unfavourable development of positive

and negative charges on the 2’- OH and the phosphate oxygen respectively
during the first step of the reaction, and the reverse charges in the second step.

Page 14 of 32
  The enzyme catalysed reaction avoids these charges by providing two histidine
residues that are capable of donating and accepting protons that neutralise
these charges .
 Acid/base catalysis depends strongly on the basic or acidic strength of the
catalytic group and its ionization state, which is determined by its pKa and the
pH the reaction takes place in.
 A strongly basic or acidic group is potentially a powerful catalyst, because it
will tend to strongly abstract or donate protons from other groups, but since its
pKa value will be far from neutral, it will rarely be in the correct protonation
state (protonated for acids and unprotonated for bases). The best acid/base
catalysts are often the weaker acid/bases, such as histidine with a pKa of
around seven, as they are more likely to be in the correct ionization state

 A phosphate ion is associated directly with the active site of the enzyme.
The amino acid residues 12 and 119 (both histidine) are nearest to the
phosphate ion.

Covalent catalysis. In covalent catalysis, the active site contains a reactive group,
usually a powerful nucleophile that becomes temporarily covalently modified in the
course of catalysis. The proteolytic enzyme chymotrypsin provides an excellent
example of thisGeneral acid-base catalysiss, a molecule other than water plays the
role of a proton donor or acceptor. Chymotrypsin uses a histidine residue as a base
catalyst to enhance the nucleophilic power of serine mechanism.

Proteases cleave proteins by a hydrolysis reaction the addition of a molecule of water

to a peptide bond chymotrypsin, cleaves peptide bonds selectively on the

carboxylterminal side of the large hydrophobic amino acids such as tryptophan,
tyrosine, phenylalanine, and methionine.

Peptide Hydrolysis by Chymotrypsin. The mechanism of peptide hydrolysis

illustrates the principles of covalent and acid-base catalysis.

Page 15 of 32
The dashed green lines indicate favorable interactions between the negatively charged
aspartate residue and the positively charged histidine residue, which make the
histidine residue a more powerful base.

Covalent Mechanism: Chymotrypsin, like many proteases follows covalent or

Double Displacement mechanism inhydrolyzes ester bonds,
in addition to peptide bonds. The hydrolysis (of peptide or ester bonds) takes
place by a two-step displacement with an amine being produced first, followed
by production of an acid.
The two steps of this double displacement mechamism are :
First step. Acylation : Formation of the acetyl-enzyme complex.
p-mitrophenylacetate (p-NPA) combines with chymotrypsin to form an enzyme-
substrate (ES) complex. The ester bond of the substrate then cleaves. One of the
products, p-nitrophenol is released from the enzyme, whereas the acetyl group
of the substrate becomes covalently attached to the enzyme.
Second step. Deacylation : Hydrolysis of the acetyl-enzyme complex.

Page 16 of 32
The second step (deacylation) is much slower than the first step (acylation), so
that it determines the overall rate of hydrolysis of esters by chymotrypsin. The
acetyl-enzyme complex is sufficiently stable to be isolated under proper
conditions. The catalytic mechanism of chymotrypsin can, thus, be
represented by,

where P1 is the amine (or alcohol) component of the substrate, E-P2 is the
covalent intermediate, and P2 is the acid component of the substrate. A distinct
feature of this mechanism is the appearance of a covalent intermediate.

In the first-step-reaction, an acetyl group is covalently bonded to the enzyme

and the group attached to chymotrypsin at E-P2 stage is an acyl group. Thus, E-
P2 is an acyl-enzyme intermediate.

LYSOZYMES

The structure of lysozyme determined by David Phillips in 1965 represented the first
three-dimensional structure for an enzyme (Figure 7.24). The protein was elliptical in

Page 17 of 32
shape (3.0 × 3.0 × 4.5 nm), with the most striking feature being a prominent cleft that
traversed one face of the molecule. Although a picture of substrate binding sites had
been established from kinetic and chemical modification studies this feature provided
a potent image of ‘active sites’.

Lysozyme contains five helical segments together with a triple stranded anti-parallel
β sheet that forms one wall of the binding site for NAG–NAM polymers and a
smaller β strand region. Models of lysozyme complexed with saccharides reveal a
cleft accommodating six units at individual sites arranged linearly along the binding
surface. The sites are designated A–F; NAM binds to sites B, D and F because there
is not enough space for the bulkier side chains at positions A, C and E. The remaining
sites are occupied by NAG.

Sites B and F accommodate NAM without distortion but at site D the sugar molecule
will only fit with distortion from its normal chair conformation into a half-chair
arrangement (Figure 7.25). Moreover, trimers of NAG bound at sites A–C are not
hydrolysed at significant rates, and by performing lysozymemediated catalysis in the
presence of H2O18 it was demonstrated that hydrolysis occurred between the C1 and
O atoms of the glycosidic bond located at subsites D and E (Figure 7.26). At this site
two side chains from Glu35 and Asp52 are located close to the C1 position of the
distorted sugar. These two side chains are the only potential catalytic groups in the
vicinity of the target C–O bond.

Glu35 is located in a non-polar environment and has an elevated pKa of ∼6.5. Asp52
in contrast has a more typical value around 3.5. The pH optimum for lysozyme is 5.0,
midway between the pKas associated with each of these side chains. Glu35, as a
result of its unusual pK, is protonated at pH 5.0 and acts as an acid catalyst donating a
proton to the oxygen atom involved in the glycosidic bond between the D and E sites
(Figure 7.27). Cleavage occurs and a portion of the substrate bound at sites E and F
can diffuse out of the cleft to be replaced by water.

The resulting cleavage leads to the formation of a carbocation which interacts with
the carboxylate group of Asp 52 (nucleophile) to form a glycosyl–enzyme
intermediate in a concerted SN2-type reaction. The carboxylate group is then
displaced from the glycosyl–enzyme intermediate by a water molecule that restores
the original configuration at the C1 centre.

Page 18 of 32
The lysozyme cleavage site after the β(1→4) linkage in alternating NAG–NAM units
of the polysaccharide

components of cell walls.

It destroys bacterial cell walls by hydrolysing the β(1→4) glycosidic bonds between
Nacetylglucosamine (NAG) and N-acetylmuramic acid (NAM) that form the bulk of
peptidoglycan cell walls . It will also hydrolyse poly-NAG chains

Page 19 of 32
Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate with NADH to form
lactate (see Section 14.3). A schematic of the enzyme’s active site is shown below;
the pyruvate is in the center

Page 20 of 32
The reaction mechanism is similar to many NADH reductions (Fig. 13–24); it is
approximately the reverse of steps 2 and 3 of Figure 14–7. The transition state
involves a strongly polarized carbonyl group of the pyruvate molecule as shown
below:

(a) A mutant form of LDH in which Arg109 is replaced with Gln shows only 5% of
the pyruvate binding and 0.07% of the activity of wild-type enzyme. Provide a
plausible explanation for the effects of this mutation.

(b) A mutant form of LDH in which Arg171 is replaced with Lys shows only 0.05% of
the wild-type level of substrate binding.

Why is this dramatic effect surprising?

(c) In the crystal structure of LDH, the guanidinium group of Arg171 and the carboxyl
group of pyruvate are aligned as shown in a co-planar “forked” configuration. Based
on this, provide a plausible explanation for the dramatic effect of substituting Arg171
with Lys.

(d) A mutant form of LDH in which Ile250 is replaced with Gln shows reduced
binding of NADH. Provide a plausible explanation for this result.

Clarke and colleagues also set out to engineer a mutant version of LDH that would
bind and reduce oxaloacetate rather than pyruvate. They made a single substitution,
replacing Gln102 with Arg; the resulting enzyme would reduce oxaloacetate to malate
and would no longer reduce pyruvate to lactate.

They had therefore converted LDH to malate dehydrogenase

Page 21 of 32
The PDH complex contains three enzymes—pyruvate dehydrogenase (E1),
dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3)—each
present in multiple copies.

The number of copies of each enzyme and therefore the size of the complex varies
among species. The PDH complex isolated from mammals is about 50 nm in
diameter—more than five times the size of an entire ribosome and big enough to be
visualized with the electron microscope .

In the bovine enzyme, 60 identical copies of E2 form a pentagonal dodecahedron (the

core) with a diameter of about 25 nm (Fig. 16–5b). (The core of the Escherichia
coli enzyme contains 24 copies of E2.)

Page 22 of 32
E2 is the point of connection for the prosthetic group lipoate, attached through an
amide bond to the e-amino group of a Lys residue . E2 has three functionally distinct
domains :
the amino-terminal lipoyl domain, containing the lipoyl-Lys residue(s);
the central E1- and E3-binding domain; and
the inner-core acyltransferase domain, which contains the acyltransferase active site.

The yeast PDH complex has a single lipoyl domain with a lipoate attached, but the
mammalian complex has two, and E. coli has three .
The domains of E2 are separated by linkers, sequences of 20 to 30 amino acid
residues, rich in Ala and Pro and interspersed with charged residues; these linkers
tend to assume their extended forms, holding the three domains apart.

The active site of E1 has bound TPP, and that of E3 has bound FAD. Also part
of the complex are two regulatory proteins, a protein kinase and a
phosphoprotein phosphatase, discussed below.

This basic E1- E2-E3 structure has been conserved during evolution
and used in a number of similar metabolic reactions, including the oxidation of
_-ketoglutarate in the citric acid cycle (described below) and the oxidation of _-
keto acids derived from the breakdown of the branched-chain amino acids
valine, isoleucine, and leucine .

Within a given species, E3 of PDH is identical to E3 of the other two enzyme

complexes.
The attachment of lipoate to the end of a Lys side chain in E2 produces a long,
flexible arm that can move from the active site of E1 to the active sites
of E2 and E3, a distance of perhaps 5 nm or more

Page 23 of 32
Page 24 of 32
the pyruvate dehydrogenase complex carries out the five consecutive reactions in the
decarboxylation and dehydrogenation of pyruvate.

Step 1 is essentially identical to the reaction catalyzed by pyruvate decarboxylase .

C-1 of pyruvate is released as CO2, and C-2, which in pyruvate has the oxidation
state of an aldehyde, is attached to TPP as a hydroxyethyl group.

This first step is the slowest and therefore limits the rate of the overall reaction. It is
also the point at which the PDH complex exercises its substrate specificity.

In step 2 the hydroxyethyl group is oxidized to the level of a carboxylic acid

(acetate). The two electronsremoved in this reaction reduce the —S—S— of a lipoyl
group on E2 to two thiol (—SH) groups. The acetyl moiety produced in this
oxidation-reduction reaction is first esterified to one of the lipoyl —SH groups, then
transesterified to CoA to form acetyl-CoA (step 3 ).

Thus the energy of oxidation drives the formation of a high-energy thioester of

acetate.
The remaining reactions catalyzed by the PDH complex (by E3, in steps 4
and 5 ) are electron transfers necessary to regenerate the oxidized (disulfide)
form of the lipoyl group of E2 to prepare the enzyme complex for another round of
oxidation.

The electrons removed from the hydroxyethyl group derived from pyruvate pass
through FAD to NAD_.

Central to the mechanism of the PDH complex are the swinging lipoyllysyl arms of
E2, which accept from E1 the two electrons and the acetyl group derived from
pyruvate, passing them to E3.

All these enzymes and coenzymes are clustered, allowing the intermediates to
react quickly without diffusing away from the surface of the enzyme complex. The
five-reaction sequence is thus an example of substrate channeling.

The intermediates of the multistep sequence never leave the complex, and the local
concentration of the substrate of E2 is kept very high.

Channeling also prevents theft of the activated acetyl group by other enzymes that
use this group as substrate.

Page 25 of 32
Oxidative decarboxylation of pyruvate to acetyl-CoA by the PDH complex.

The fate of pyruvate is traced in red.

In step 1 pyruvate reacts with the bound thiamine pyrophosphate (TPP) of pyruvate
dehydrogenase (E1), undergoing decarboxylation to the hydroxyethyl derivative .

Pyruvate dehydrogenase also carries out step 2 , the transfer of two electrons and
the acetyl group from TPP to the oxidized form of the lipoyllysyl group of the core
enzyme, dihydrolipoyl transacetylase (E2), to form the acetyl thioester of the reduced
lipoyl group.

Step 3 is a transesterification in which the —SH group of CoA replaces the —

SH group of E2 to yield acetyl-CoA and the fully reduced (dithiol) form of the
lipoyl group.

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 In step 4 dihydrolipoyl dehydrogenase (E3) promotes transfer of two hydrogen
atoms from the reduced lipoyl groups of E2 to the FAD prosthetic group of E3,
restoring the oxidized form of the lipoyllysyl group of E2.

In step 5 the reduced FADH2 of E3 transfers a hydride ion to NAD_, forming

NADH

Oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase complex.

Lipoic acid is joined by an amide link to a lysine residue of the transacetylase

component of the enzyme complex. It forms a long flexible arm, allowing the
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lipoic acid prosthetic group to rotate sequentially between the active sites of
each of the enzymes of the complex.

(NAD+, nicotinamide adenine dinucleotide; FAD, flavin adenine dinucleotide;

TDP, thiamine diphosphate.)

Pyruvate kinase, the enzyme catalyzing the third irreversible step in glycolysis,
controls the outflow from this pathway.

This final step yields ATP and pyruvate, a central metabolic intermediate that can be
oxidized further or used as a building block. Several isozymic forms of pyruvate
kinase (a tetramer of 57-kd subunits) encoded by different genes are present in
mammals: the L type predominates in liver, and the M type in muscle and brain.

The L and M forms of pyruvate kinase have many properties in common. Both bind
phosphoenolpyruvate cooperatively. Fructose 1,6-bisphosphate, the product of the
preceding irreversible step in glycolysis, activates both isozymes to enable them to
keep pace with the oncoming high flux of intermediates. ATP allosterically inhibits
both the L and the M forms of pyruvate kinase to slow glycolysis when the energy
charge is high. Finally, alanine also allosterically inhibits the pyruvate kinases

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Superoxide dismutase. This enzyme scavenges superoxide radicals by
catalyzing the conversion of two of these radicals into hydrogen peroxide and
molecular oxygen.

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The oxidized form of the enzyme is reduced by superoxide to form oxygen. The
reduced form of the enzyme, formed in this reaction, then reacts with a second
superoxide ion to form peroxide, which takes up two protons along the reaction
path to yield hydrogen peroxide

the transfer of a single electron to O 2 forms superoxide anion, whereas the

transfer of two electrons yields peroxide.

Explain the catalytic role of antibodies with an example.

 Antibodies are immunoglobulins, which, are proteins. Catalytic

antibodies
are antibodies with catalytic activity (catalytic antibodies are also called
abzymes.
 Like other antibodies, catalytic antibodies are elicited in an organism in
response to immunological challenge by a foreign molecule called an
antigen.

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  In this case, the antigen is purposefully engineered to be an analog of
the transition state in a reaction.
 The rationale is that a protein specific for binding the transition state of a
reaction will promote entry of the normal reactant into the reactive,
transition state conformation.
Thus, a catalytic antibody facilitates, or catalyzes, a reaction by forcing
the conformation of its substrate in the direction of its transition state.
 An example of this principle has been to prepare ester analogs by
substituting a phosphorus atom for the carbon in the ester group .

 The phosphonate compound mimics the natural transition state of ester

hydrolysis, and antibodies elicited against these analogs act like enzymes
in accelerating the rate of ester hydrolysis as much as 1000-fold.

 Abzymes have been developed for a number of other classes of reactions,

including COC bond formation via aldol condensation and the pyridoxal
5-P–dependent aminotransferase reaction .
 Catalytic antibodies apparently occur naturally. Autoimmune diseases are
diseases that arise because an individual begins to produce antibodies
against one of their own cellular constituents.
 Multiple sclerosis (MS), one such autoimmune disease, is characterized
by gradual destruction of the myelin sheath surrounding neurons
throughout the brain and spinal cord.
 Blood serum obtained from some MS patients contains antibodies
capable of carrying out the proteolytic destruction of myelin basic protein
(MBP).

 That is, these antibodies were MBP-destructive proteases. Thus, some

antibodies may be proteases.

(a) The intramolecular hydrolysis of a hydroxy ester to yield as products a _-lactone and the
alcohol phenol. Note the cyclic transition state. (b) The cyclic phosphonate ester analog of the cyclic transition

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