Chap.

6B Enzymes
Introduction to Enzymes
How Enzymes Work
Enzyme Kinetics as an Approach to
Understanding Mechanism
Examples of Enzymatic Reactions
Regulatory Enzymes

Fig. 6-22. The chymotrypsin

enzyme-substrate complex.

Intro. to Enzyme Mechanisms

An understanding of the complete mechanism of action of a purified
enzyme requires identification of all substrates, cofactors,
products, and regulators. Moreover, it requires a knowledge of 1)
the temporal sequence in which enzyme-bound reaction
intermediates form, 2) the structure of each intermediate and
each transition state, 3) the rates of interconversion between
intermediates, 4) the structural relationship of the enzyme to each
intermediate, and 5) the energy contributed by all reacting and
interacting groups to intermediate complexes and transition states.
As yet, there is probably no enzyme for which all of these
features are completely known. However, many of these
mechanistic aspects have been established for enzymes such as
chymotrypsin, which will be the focus of our discussion of enzyme
mechanisms.

The Structure of Chymotrypsin (CT)

Bovine pancreatic chymotrypsin (Mr
25,191) is a protease, or enzyme
that catalyzes the hydrolytic
cleavage of peptide bonds in target
proteins. CT is specific for peptide
bonds adjacent to aromatic amino
acid residues (Trp, Phe, & Tyr).
Cleavage occurs on the C-terminal
side of these residues. The threedimensional structure of CT is shown
in Fig. 6-19, with functional groups in
the active site emphasized. The
protein consists of three polypeptide
chains linked by disulfide bonds. The
three chains are derived by
proteolytic processing of a slightly
longer preprotein known as
chymotrypsinogen, as explained in Fig.
6-38, below). The key catalytic
residues of the active site (His57,
Asp102, Gly193, and Ser195 of the
primary structure) are brought
together in three-dimensional space
in the tertiary structure. The
hydrophobic pocket in which the
aromatic amino acid side-chain of the
substrate is bound is highlighted in

Intro. to the CT Reaction Mechanism (I)

The reaction catalyzed by CT illustrates the principle of transition
state stabilization and also provides a classic example of general
acid-base catalysis and covalent catalysis. CT enhances the rate of
peptide bond hydrolysis by a factor of at least 10 9. It does not
catalyze a direct attack of water on the peptide bond. Instead, a
transient covalent acyl-enzyme intermediate is formed. The
reaction has two distinct phases. In the acylation phase, the
peptide bond is cleaved and an ester linkage is formed between the
peptide carbonyl carbon and the enzyme. In the deacylation phase,
the ester linkage is hydrolyzed and the nonacylated enzyme is
regenerated. As covered below, the nucleophile in the acylation
phase is the oxygen atom in the side-chain of Ser195. Proteases
with serine residues that play this role in reaction mechanisms are
called serine proteases. The pKa of a serine hydroxyl group is
generally too high for the unprotonated form to be present in
significant concentrations at physiological pH. However, in CT,
Ser195 is linked to His57 and Asp102 in a hydrogen-bonding network
referred to as the catalytic triad. The association of His57 and
Asp102 raises the pKa of the His residue to > 12, allowing the His
to act as an enhanced general base that can remove the proton
from Ser195 creating an extremely reactive nucleophile. At later
reaction stages, His57 acts as a proton donor, protonating the
amino group in the displaced portion of the substrate (the leaving
group).

Intro. to the CT Reaction Mechanism (II)

As the Ser195 oxygen attacks the carbonyl group of the substrate,
a very short-lived tetrahedral intermediate is formed in which the
carbonyl oxygen acquires a negative charge (Fig. 6-22, Step 2).
This charge, forming within a pocket on the enzyme called the
oxyanion hole, is stabilized by hydrogen bonds to the CT backbone.
One of these hydrogen bonds is contributed by Gly193 and is present
only in this intermediate and in the transition state for its
formation and breakdown. This interaction reduces the activation
energy required to reach these states. This is an example of the
use of binding energy in catalysis.

Evidence for an Acyl-enzyme Intermediate

The first evidence for a covalent acylenzyme intermediate came from a

classic application of pre-steady state
kinetics. In addition to its action on
polypeptides, CT also catalyzes the
hydrolysis of small esters and amides.
These reactions are much slower than
peptide hydrolysis because less binding
energy is available with smaller
substrates, and they are therefore
easier to study. CT hydrolysis of the
ester p-nitrophenylacetate, as
measured by the release of pnitrophenol, proceeds with a rapid burst
before leveling off to a slower rate
(Fig. 6-20). By extrapolating back to
zero time, the burst phase corresponds
to just under one molecule of pnitrophenol released for every enzyme
molecule present. This is consistent with
the rapid acylation of all enzyme
molecules, with the rate for subsequent
turnover of the enzyme limited by a
slower deacylation step.

pH Dependence of the CT Reaction

The rate of CT-mediated cleavage produces a bell-shaped pH-rate

profile with an optimum at pH 8.0 (Fig. 6-21). The rate (v) being
plotted is that at low substrate concentrations and thus reflects
the term kcat/Km. The plot can be broken down to its components
using kinetic methods to determine the terms kcat and Km separately
at each pH (not discussed). When this is done (Parts b & c), it
becomes clear that the transition just above pH 7 is due to changes
in kcat, whereas the transition above pH 8.5 is due to changes in
1/Km. Kinetic and structural studies have shown that the transitions
illustrated in Parts (b) and (c) reflect the ionization states of the
His57 side-chain (when substrate is not bound) and the -amino
group of Ile16 at the amino terminus of the B chain, respectively.
For optimal activity, His57 must be unprotonated so it can remove
the hydrogen atom from Ser195 at the start of the CT reaction.
Ile16 must be protonated for binding of the substrate, hence the
effect on 1/Km.

CT Mechanism: Acylation Phase (Step 1)

The next few slides cover the acylation and deacylation phases of
the CT reaction mechanism. These slides are derived from Fig. 622.

CT Mechanism: Acylation Phase (Step 2)

CT Mechanism: Acylation Phase (Step 3)

CT Mechanism: Acylation Phase (Step 4)

CT Mechanism: Deacylation Phase (Step 5)

CT Mechanism: Deacylation Phase (Step 6)

CT Mechanism: Deacylation Phase (Step 7)

Review of Nucleophiles and Electrophiles in

Biochemistry
In many reactions of biochemistry, an electron-rich atom (a
nucleophile) reacts with an electron-deficient atom (an electrophile).
Some common nucleophiles and electrophiles in biochemistry are
shown in the diagram below.

Intro. to Regulatory Enzymes

Regulatory enzymes exhibit increased or decreased catalytic

activity in response to certain signals. Regulatory enzymes play
important roles in governing the rate of flux of compounds through
metabolic pathways in cells. The activities of regulatory enzymes
are modulated in a number of ways. Allosteric enzymes are
regulated by the reversible, noncovalent binding of regulatory
molecules called allosteric modulators or effectors. Another type
of regulation occurs via reversible covalent modification of enzyme
side-chains. Both types of regulatory enzymes tend to be
multisubunit proteins. In many cases the regulatory sites and the
active site are on separate subunits. Two other types of
regulation also are common. These are regulation achieved by the
binding of a separate regulatory protein to the enzyme of
interest, and proteolytic cleavage of the target enzyme, removing
peptide segments that inhibit its activity. Unlike effectormediated regulation, regulation by proteolytic cleavage is
irreversible. Note that more than one type of regulation (e.g.,
allosteric and covalent) can occur for a single regulatory enzyme.

Conformational Changes in Allosteric

Regulatory Enzymes

The modulators of an allosteric enzyme may be inhibitory or

stimulatory. If the modulator and substrate are the same the
regulation is called homotropic. If the modulator and substrate
are different molecules the regulation is called heterotropic.
Allosteric enzymes generally have one or more regulatory, or
allosteric, sites for binding the modulator. Just as an enzymes
active site is specific for its substrate, each regulatory site is
specific for its modulator. In many allosteric enzymes, the
substrate-binding and modulator-binding sites(s) are on different
subunits. These are called the catalytic (C) and regulatory (R)
subunits. In Fig. 6-32, binding of the
positive (stimulatory) modulator (M) to
its specific site on the regulatory
subunit is communicated to the catalytic
subunit through a conformational
change. This change renders the
catalytic subunit active and capable of
binding the substrate (S) with higher
affinity. On dissociation of the
modulator from the regulatory subunit,
the enzyme reverts to its inactive or
less active form

Regulation of Aspartate Transcarbamoylase

(I)
A classic example of a regulatory enzyme is aspartate
transcarbamoylase (ATCase). This enzyme catalyzes a regulatory
step early in the biosynthesis of pyrimidine nucleotides. In this
reaction, carbamoyl phosphate and aspartate combine to form Ncarbamoyl aspartate.

Regulation of Aspartate Transcarbamoylase

(II)
ATCase has 12 polypeptide chains organized into 6 catalytic
(organized as 2 trimeric complexes) and 6 regulatory (organized as
3 dimeric complexes) subunits (Fig. 6-33). The regulatory subunits
have binding sites for ATP and CTP, which function as positive and
negative regulators, respectively. CTP is one of the end products
of the pyrimidine nucleotide biosynthetic pathway, and negative
regulation by CTP serves to limit ATCase activity under conditions
when CTP is abundant. High concentrations of ATP indicate that
cellular metabolism is robust, the cell is growing, and additional
pyrimidine nucleotides may be needed to support RNA transcription
and DNA replication. As shown in the diagram, the binding of the
negative modulator CTP to the regulatory subunits produces large
changes in enzyme conformation, and converts the complex to the
less active T state.

Kinetic Properties of Regulatory Enzymes (I)

Allosteric enzymes show relationships between V0 and [S] that
differ from MM kinetics. They do exhibit saturation with the
substrate when [S] is sufficiently high, but for allosteric enzymes,
plots of V0 versus [S] usually produce a sigmoid saturation curve,
rather than the hyperbolic curve typical of nonregulatory enzymes.
On the sigmoid saturation curve, a value of [S] at which the V 0 is
half-maximal can be identified, but it cannot be referred to as the
Km because the enzyme does not follow the hyperbolic MM
relationship. Instead the symbol [S]0.5 or K0.5 is often used to
represent the substrate concentration giving half-maximal velocity
for the reaction catalyzed by an allosteric enzyme.
Sigmoid kinetic behavior generally reflects cooperative interactions
between multiple protein subunits. Changes in the structure of one
subunit are translated into structural changes in adjacent subunits
due to changes in noncovalent interactions at the interfaces
between subunits. Sigmoid kinetic behavior is explained by the
concerted and sequential models for subunit interactions (Fig. 515).

Kinetic Properties of Regulatory Enzymes

(II)
Substrate-activity curves for
representative allosteric enzymes that
show complex responses to their
modulators are shown in Fig. 6-34. In
Panel (a), the sigmoid curve is for a
homotropic enzyme, for which the
substrate also serves as a positive
modulator. This curve (black)
resembles the oxygen-saturation curve
for hemoglobin (Fig. 5-12). The
sigmoidal curve is a hybrid curve in
which the enzyme is present primarily
in the relatively inactive T state at
low substrate concentration, and
primarily in the more active R state
at high substrate concentration. The
curves for pure T and R states are
plotted separately in color. ATCase
exhibits a kinetic pattern similar to
this in response to the concentrations
of its substrates, aspartate and
carbamoyl phosphate.

Kinetic Properties of Regulatory Enzymes

(III)
In Panel (b) of Fig. 6-34, curves
are plotted showing the effects of
several different concentrations of a
heterotropic positive modulator (+)
and a heterotropic negative
modulator (-) on an allosteric
enzyme in which K0.5 is altered
without a change in Vmax. The
central curve shows the substrateactivity relationship in the absence
of modulators. The curves shown are
similar for ATCase in the presence
of the negative modulator CTP and
the positive modulator ATP.

Kinetic Properties of Regulatory Enzymes

(IV)
In Panel (c) of Fig. 6-34, curves are plotted showing the effects
of modulators that change Vmax without significantly changing K0.5.
This is a less common type of allosteric regulation, and it is not
observed for ATCase.

Regulation by Reversible
Covalent Modification

For many enzymes, activity also is regulated

by reversible covalent modification of one or
more amino acid residues in the enzyme. Over
500 different types of covalent modifications
have been found in proteins. Common modifying
groups are shown in Fig. 6-35 and include
phosphorylation, adenylylation, methylation,
myristoylation, and ubiquitination. In the case
of ubiquitination, the small protein ubiquitin is
attached to a lysine residue in the target
protein flagging it for proteolytic destruction.
The modifying groups are usually attached to a
regulated enzyme by a separate modifying
enzyme (e.g., a methylase in the case of
methylation). Introduction of the modifying
group alters the local properties of the enzyme
which typically undergoes a change in
conformation and activity. Introduction of a
hydrophobic myristoyl group triggers the
association of the protein with a membrane.
Phosphorylation is the most important type of
covalent modification, and it is discussed
further in subsequent slides.

Regulation by Reversible Phosphorylation:

Muscle Glycogen Phosphorylase (I)
It is estimated that one-third of all proteins in a eukaryotic cell
are phosphorylated, and one, or often many phosphorylation
events are part of virtually every regulatory process. Some
proteins have only one phosphorylation site, whereas others have
several, and a few have dozens of sites for phosphorylation. The
attachment of phosphoryl groups to specific amino acid residues
of a protein is catalyzed by enzymes called protein kinases. In
these reactions, typically the -phosphoryl group from a
nucleoside triphosphate (usually ATP) is transferred to a
particular Ser, Thr, Tyr, or occasionally His residue in the
target protein. Removal of the same phosphoryl group from the
protein is performed by enzymes called protein phosphatases. The
introduced phosphoryl group can cause major changes in the
conformation of the modified enzyme by virtue of steric effects,
or hydrogen bonding and ionic interactions to neighboring
residues. This can lead to effects on substrate binding and
catalysis.

Regulation by Reversible Phosphorylation:

Muscle Glycogen Phosphorylase (II)

An important example of enzyme regulation by phosphorylation is

seen with glycogen phosphorylase of muscle and liver. This enzyme
catalyzes the breakdown of glycogen stores via the reaction
Glycogen(n) + Pi glycogen(n-1) + glucose 1-P

In the liver, the released glucose 1-P is typically converted to

free glucose and released to the bloodstream. In skeletal muscle,
glucose 1-P is metabolized via the glycolytic pathway for energy
production.
Glycogen phosphorylase is a homodimeric enzyme that exists in two
forms--the less active phosphorylase b form and the more active
phosphorylase a species (Fig. 6-36). Activation by phosphorylation
is catalyzed by the enzyme phosphorylase kinase which attaches
phosphate groups to serine residues
(Ser14) located near the N-termini of
both subunits. The enzyme phosphoprotein
phosphatase 1 (PP1) removes these two
phosphates by hydrolysis, inactivating the
enzyme. The phosphorylation of Ser14
causes major structural changes near the
N-termini of the glycogen phosphorylase
chains that produce a more active
enzyme. Note that glycogen phosphorylase
is also regulated by noncovalent allosteric
modulation (Fig. 6-42, not covered).

Multiple Phosphorylations Allow Exquisite

Regulatory Control

The Ser, Thr, and Tyr residues that are typically phosphorylated
in regulatory proteins occur within common structural motifs, called
consensus sequences, that are recognized by specific protein
kinases. For example the important kinase, protein kinase A,
recognizes Ser/Thr residues in the consensus sequence -x-R-[RK]x-[ST]-B-, where B is any hydrophobic amino acid (Table 6-10,
not shown). Some proteins, such as glycogen synthase (Fig. 6-37),
have consensus sequences recognized by several different protein
kinases. In some cases, phosphorylation is hierarchical: a certain
residue can only be phosphorylated if a neighboring residue has
already been phosphorylated. Glycogen
synthase, for example, is not a substrate
for glycogen synthase kinase 3 until one
site has been phosphorylated by casein
kinase II. Some phosphorylations inhibit
glycogen synthase more than others, and
some combinations of phosphorylations
have a cumulative effect on activity. The
multiple regulatory phosphorylations
provide the potential for finely tuned
modulation of enzyme activity by
controlling signals, such as hormones.

Regulation By Proteolytic Cleavage

A number of proteolytic enzymes are synthesized in inactive forms
called zymogens to control their catalytic activity until needed.
When activity is needed they are then cleaved by an activating
protease. Many proteolytic enzymes of the pancreas and stomach
are regulated this way. For example, chymotrypsin and trypsin are
initially synthesized as the zymogens chymotrypsinogen and
trypsinogen (Fig. 6-38). Specific cleavages remove residues from
these zymogens and expose the active sites. Because cleavage is
irreversible, other mechanisms are needed to inactivate these
enzymes. For example, proteases such as trypsin are inactivated
by the tight binding of the
inactivator protein, pancreatic
trypsin inhibitor. Proteases are
not the only proteins activated
by proteolysis. Such other
proteins are not called
zymogens, but instead are called
proproteins or proenzymes. For
example, collagen is initially
synthesized as the soluble
precursor procollagen which is
proteolytically processed to
collagen chains.