Biochemistry

PRMSU, 1st Semester ST 2020-2021

College of Education

Module 3: Enzymes
This module covers the following topics:

A. Properties of Enymes

B. Role of Coenzymes

C. Classification of Enzymes

D. Enzyme Mechanism

E. Enzyme Kinetics

F. Enzyme Inhibition

G. Factors Affecting the Rate of Catalytic Action

At the end of this module, the students are expected to:

 Describe how enymes works;
 List the major enzyme categories;
 Name an enzyme, given the type of reaction that it catalyzes;
 Explain enzyme kinetics and how it provides information about the behavior of
enzymes;
 Understand the role of enzymes and the mechanisms of their actions in various
biochemical and metabolic pathways;
 Differentiate the various types of enzyme inhibition; and
 Identify the factors that affect enzyme activity.

INTRODUCTION:
While you are reading this module, several processes are simultaneously taking place in
your body. New tissues are made to replace the old, food is converted to energy, and substances
that the body does not need are disposed. All these processes involve thousands of complex
chemical reactions.

The living cell is like a factory of nonstop biochemical activities. The sum total oif these
activities is what is known as metabolism. What is remarkable about this is that the reactions

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take place in mild conditions of pH (our physiological pH is 7.35) and temperature (our normal
body temperature is 370C). In the laboratory, a researcher can break down an average protein by
boiling it for 24 hours in a strong acid solution, e.g. HCl. In the body, however, complete
breakdown of a protein can take about four hours. It facilitates us to think that the living cell
performs with such speed and precision amultitude of chemical reactions that otherwise occur
with immeasurable slowness at the same temperature and pressure.

The secret lies in the remarkable catalytic proteins, the ENZYMES, that are found in the
living cell. Enzymes enable these complex chemical reactions to take place in our body. By
nature enzymes are proteins and as a special type of protein, it is very essential in catalysis.
Enzymes are the catalysts of biochemical systems that direct all metabolic events. Biochemical
reactions involve exchange of electrons between atoms of reacting molecules. Nearly all known
enzymes are proteins. It has also been recently verified that several RNA molecules do perform
enzymatic activities. Some enzymes work alone, others work concertedly with simple inorganic
ions, or with simple organic systems known as coenzymes.

This module will focus on this important group of proteins. Aside from the properties and
classification of enzymes, the kinetics involved and the inhibition of enzyme activity will also be
covered by the discussion.

DISCUSSION AND EXAMPLES:

A. PROPERTIES OF ENZYMES

Enzymes are the agents of metabolic function. Acting in sequence, enzymes form
metabolic pathways by which nutrient molecules are degraded, energy is released and converted
into metabolically useful forms, and precursors are generated and transformed to create the
literally thousands of distinctive biomolecules found in any living cell. Situated at key junctions
of metabolic pathways are specialized regulatory enzymes capable of sensing the momentary
metabolic needs of the cell and adjusting their catalytic rates accordingly. The responses of these
enzymes ensure the harmonious integration of the diverse and often divergent metabolic
activities of cells so that the living state is promoted and preserved.

Enzymes are complex biological molecules, primarily or entirely protein, which behave
as biological catalysts. As catalysts, they alter the rate of a chemical reaction without themselves
being consumed in the reaction. Enzymes are normally very specific in their action, often
targeting only one specific reacting species, known as the substrate.

This specificity includes stereospecificity, the arrangement of the substrate atoms in

three-dimensional space. Stereospecificity is illustrated by the fact that if the D-glucose in your
diet were replaced by its enantiomer, L-glucose, you would not be able to metabolize this
otherwise identical enantiomer.

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Enzymes occur in many forms. Some enzymes consist entirely of proteins, whereas
others have non-protein portions known as cofactors. The cofactor may be a metal ion, such as
magnesium, or an organic substance. We call an organic cofactor a coenzyme (there is no
specific term for a metallic cofactor). An enzyme lacking its cofactor is an apoenzyme, and the
combination of an apoenzyme and its cofactor is a holoenzyme. A metalloenzyme contains an
apoenzyme and a metal ion cofactor. A tightly bound coenzyme is a prosthetic group. (Wow! We
know that this is a lot of terminology, but hang in there. The key is the enzyme.)

One region on the enzyme, the active site, is directly responsible for interacting with the
reacting molecule(s). When a reacting molecule, the substrate, binds to this active site, a reaction
may occur. Other materials besides the enzyme and substrate, may be necessary for the reaction
to occur.

The ACTIVE SITE is a specialized region of the protein where the enzymes interacts
with the substrate. The active site of an enzyme is generally a pocket or cleft that is specialized
to recognize specific substrates and catalyze chemical transformations. It is formed in the three-
dimensional structure by a collection of different amino acids (active-site residues) that may or
may not be adjacent in the primary sequence. The interactions between the active site and the
substrate occur via the same forces that stabilize protein structure: hydrophobic interactions,
electrostatic interactions (charge–charge), hydrogen bonding, and van der Waals interactions.
Enzyme active sites do not simply bind substrates; they also provide catalytic groups to facilitate
the chemistry and provide specific interactions that stabilize the formation of the transition state
for the chemical reaction.

In many cases, the cell initially produces the enzyme in an inactive form called a
proenzyme or zymogen, which must undergo activation for it to function. The enzyme trypsin
illustrates why it is sometimes necessary to generate an inactive form of an enzyme. Trypsin is
one of the enzymes present in the stomach that is responsible for the digestion of proteins. Its
production, as an inactive form, occurs in the cells of the stomach walls, and activation occurs
after its release into the stomach. If trypsin were produced in the active form, it would
immediately proceed to begin digesting the cell that produced it. Eating yourself is not a good
thing.

The activation of the inactive form of an enzyme serves as one form of enzyme control.
Inhibition is another method of enzyme control. The two general types of inhibition are
competitive inhibition and noncompetitive inhibition. In competitive inhibition, another species
competes with the substrate to interact with the active site on the enzyme. In noncompetitive
inhibition, the other species binds to some site other than the active site. This binding alters the
overall structure of the enzyme so that it no longer functions as a catalyst.

Enzymes are characterized by three distinctive features: specificity, catalytic powers,

and regulation.

THE SPECIFICITY OF ENZYMATIC ACTIONS

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Enzymes are usually impressively specific in their action. The specificity toward
substrate is sometimes almost absolute. For many years urea was believed to be the only
substrate for the enzyme urease and succinate the only substrate for succinate dehydrogenase.
Even after much searching for other substrates, only one or two closely related compounds could
be found that were acted on at all. In other cases enzymes can use a class of compounds as
substrates. For example, the D-amino acid oxidase of kidney oxidizes a variety of D-amino
acids but does not touch L-amino acids.

Almost as impressive as the substrate specificity of enzymes is the specificity for a given
type of reaction. Many substrates are capable of undergoing a variety of different chemical
reactions, either unimolecular or with water or some other compound present in the cell. The
enzyme catalyzes only one of these reactions.

Although side reactions may occur to a small extent, the most impressive thing in
comparing an enzymecatalyzed reaction with an uncatalyzed organic reaction is that the latter
often produces large amounts of side reaction products, but the enzymatic reaction does not.

REASONS WHY ENZYMATIC REACTIONS ARE VERY SPECIFIC

1. Complementarity of Substrate and Enzyme Surfaces

Impressed by the specificity of enzymatic action, biochemists early adopted a “lock-and-

key” theory which stated that for a reaction to occur the substrate must fit into an active site
precisely. Modern experiments have amply verified the idea. A vast amount of kinetic data on
families of substrates and related competitive inhibitors support the idea and numerous X-ray
structures of enzymes with bound inhibitors or with very slow substrates have given visual
evidence of the reality of the lock-and-key concept. Directed mutation of genes of many
enzymes of known threedimensional structure has provided additional proof.

As anticipated, hydrophobic groups of substrates or inhibitors usually contact

hydrophobic regions of the protein and the fit is tight. For example, the active site of
chymotrypsin contains a “hydrophobic pocket” designed to hold a large hydrophobic side chain,
thus providing the specificity observed with this enzyme. Likewise, polar groups of substrates
contact polar groups of the enzyme. The interactions are complementary, positive charges fitting
against negative and with correctly formed hydrogen bonds. Trypsin, whose structure is similar
to that of chymotrypsin, exerts its specificity for a positively charged side chain next to the bond
cleaved by virtue of the presence of a negatively charged carboxylate at the bottom of the
hydrophobic pocket. Several C = O and N– H groups of the peptide linkages in the substrate
form hydrogen bonds to the edge of a β sheet in the protein, in effect making the substrate an
added β strand in the sheet. Aspartate aminotransferase acts on the dicarboxylic amino acids
glutamate and aspartate. A pair of arginine side chains bind the two carboxylates of the substrate
while the – NH3+ of the substrate is attracted to a negatively charged group in the coenzyme
pyridoxal phosphate, which is also present in the active site.

2. Stereospecificity and Prochiral Centers

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Most enzymes possess an infallible ability to recognize the difference between the right
side and the left side of an organic substrate even when the latter has perfect bilateral symmetry.
In fact, this ability is limited to prochiral centers of molecules and is a natural consequence of
their reaction with the chiral enzyme. Consider carbon atom number 1 of ethanol. The two
attached hydrogen atoms are chemically identical and would react identically with a nonchiral
reagent. Nevertheless, these atoms are no more equivalent stereochemically than are your right
and your left arms. We say that the molecule has a prochiral center at C-1. A prochiral center on
a tetrahedrally bonded carbon atom always contains two identical atoms or groups but a total of
three different kinds of atoms or groups. If the priority of this H is elevated,m e.g., by
substitution of 2H for 1H the configuration would be R (the priority of this 2H would become c).

The two hydrogen atoms on C-1 of ethanol are called enantiotopic because replacement
of one or the other by a fourth different kind of atom or group would produce a pair of
enantiomers. This fact suggests a way of naming the positions occupied by two enantiotopic
atoms or groups.

The ability of an enzyme to recognize a single hydrogen of a pair of hydrogens on a CH 2

group was at first a surprise to many biochemists. However, it is a natural result of the
complementarity of enzyme and substrate surfaces, just as the fit of a shoe is determined by the
complementarity of surfaces of foot and shoe. Only a chiral catalyst can have this ability.

3. Induced Fit and Conformational Changes

Results of many X-ray studies indicate that the lock-and-key picture of enzyme action
must be modified. If an enzyme is a “lock” and the substrate the “key,” the entrance of the key
into the lock often induces a conformational change in the protein. Binding of substrates may be
imperfect in the initially formed ES complex but may be more nearly perfect a few nanoseconds
or microseconds later as the protein readjusts its structure to accommodate the substrate. The
substrate has induced a fit. For example, when an amino acid substrate binds to aspartate
aminotransferase one whole domain of the enzyme moves inward, packing hydrophobic side
chains of the protein against the substrate. This strengthens the electrostatic interactions between
the ion pairs that orient the substrate and align it for reaction. Similar conformational changes
have been observed for citrate synthase, glycogen phosphorylase, various kinases, alcohol
dehydrogenase, and a growing list of other enzymes. Accompanying changes in circular
dichroism, ultraviolet spectra, and sedimentation constants are often observed.

4. Specificity and kcat

Enzyme specificity is often observed not only in binding but also in the rate at which ES
is converted to products. Thus, it is the values of kcat/ Km that determine specificity. Good
examples are provided by chymotrypsin and related serine proteases, 2 for which substrates with
the shortest chains are often bound as well as those with longer chains but react more slowly. For
example, N-acetylphenylalanine amide binds to chymotrypsin about as well as does the longer N-
acetylphenylalanylalanine amide but reacts only 1/47 as fast.2 One might anticipate that
increasing the length of the substrate would make it bind more tightly because of the greater
number of contacts between substrate and enzyme. It has often been suggested that the reason

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that this does not happen is that thebinding of the longer, more specific substrates distorts the
enzyme and that the binding energy is now stored in the enzyme. It is as if the enzyme contained
an internal spring which would be compressed when the substrate binds. This would keep the
binding weak but the energy in the spring might then be used to increase the velocity.

5. Proofreading
Although enzymes may be very specific they do make mistakes. This is of particular
concern for processes such as protein synthesis in which the correct amino acid is placed at each
position in the sequence with an error rate that has been estimated for E. coli as only 1 in 104. It
would be impossible for an enzyme designed to attach valine to its specific transfer RNA to
avoid attaching the smaller alanine if discrimination between the two were based solely on the
Gibbs energy differences of binding.103 However, it would be easier for an enzyme to exclude
larger amino acids. This problem may be resolved by use of multistep screening. For example,
isoleucyl-tRNA synthetase does occasionally attach the smaller valine to the specific tRNA,Ile
but when it does the enzyme in a “proofreading and editing” step hydrolyzes off the incorrect
amino acid. The active site for this hydrolyzing activity, whether at a different place on the
enzyme surface or created by a conformational change, may be able to exclude sterically the
larger isoleucyl residue while acting on the valyl-tRNA. This editing mechanism for isoleucyl-
tRNA synthetase was demonstrated directly in 1998 by X-ray crystallography on complexes of
the enzyme with L-isoleucine and L-valine. Both substrates fit into the ATP-requiring synthetic
site but neither isoleucine nor isoleucyltRNA will fit into the editing site which is located in an
adjacent β-barrel domain. Proofreading steps based on differing chemical properties as well as
size can also be visualized.

CATALYTIC POWER

Enzymes display enormous catalytic power, accelerating reaction rates as much as 10 16

over uncatalyzed levels, which is far greater than any synthetic catalyts can achieve, and
enzymes accomplish these astounding feats in dilute aqueous solutions under mild conditions of
temperature and pH.

REGULATION

Regulation of enzyme activity is achieved in a variety of ways, ranging from controls

over the amount of enzyme protein produced by the cell to more rapid, reversible interactions of
the enzyme with metabolic inhibitors and activators. This module is devoted to discussions of
enzyme regulation. Because most enzymes are proteins, we can anticipate that the functional
attributes of enzymes are due to the remarkable versatility found in protein structures.

B. ENZYME NOMENCLATURE

Traditionally, enzymes often were named by adding the suffix -ase to the name of the
substrate upon which they acted, as in urease for the urea-hydrolyzing enzyme or phosphatase
for enzymes hydrolyzing phosphoryl groups from organic phosphate compounds. Other enzymes
acquired names bearing little resemblance to their activity, such as the peroxide-decomposing

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enzyme catalase or the proteolytic enzymes (proteases) of the digestive tract, trypsin and pepsin.
Because of the confusion that arose from these trivial designations, an International Commission
on Enzymes was established in 1956 to create a systematic basis for enzyme nomenclature.
Although common names for many enzymes remain in use, all enzymes now are classified and
formally named according to the reaction they catalyze. Six classes of reactions are recognized.
Within each class are subclasses, and under each subclass are subsubclasses within which
individual enzymes are listed. Classes, subclasses, subsubclasses, and individual entries are each
numbered, so that a series of four numbers serves to specify a particular enzyme. A systematic
name, descriptive of the reaction, is also assigned to each entry. To illustrate, consider the
enzyme that catalyzes this reaction:

ATP + D-glucose ADP + D-glucose-6-phosphate

A phosphate group is transferred from ATP to the C-6-OH group of glucose, so the
enzyme is a transferase (Class 2, Table 1). Subclass 7 of transferases is enzymes transferring
phosphorus-containing groups, and sub-subclass 1 covers those phosphotransferases with an
alcohol group as an acceptor. Entry 2 in this sub-subclass is ATP: D-glucose-6-
phosphotransferase, and its classification number is 2.7.1.2. In use, this number is written
preceded by the letters E.C., denoting the Enzyme Commission. For example, entry 1 in the
same sub-subclass is E.C.2.7.1.1, ATP: D-hexose-6-phosphotransferase, an ATP-dependent
enzyme that transfers a phosphate to the 6-OH of hexoses (that is, it is nonspecific regarding its
hexose acceptor). These designations can be cumbersome, so in everyday usage, trivial names
are employed frequently. The glucose-specific enzyme, E.C.2.7.1.2, is called glucokinase and the
nonspecific E.C.2.7.1.1 is known as hexokinase. Kinase is a trivial term for enzymes that are
ATP-dependent phosphotransferases.

C. CLASSIFICATION OF ENZYMES

Ever wonder who gets to name chemicals? Well, the answer varies, but for enzymes it’s
the Enzyme Commission of the International Union of Biochemistry (IUB) that’s responsible.
Common names for enzymes begin with some description of its action plus an -ase suffix.
(Enzymes that were named before the implementation of the -ase system, such as trypsin, do not
follow this convention.) The Enzyme Commission has also developed a numerical system for
classifying enzymes. The names begin with EC, for Enzyme Commission, and end with four
numbers, separated by decimal points, describing the enzyme. An example of this nomenclature
is EC 2.7.4.4.

The first number in the EC name refers to the major enzyme class, and there are six
major enzyme classes, summarized in Table 6-1. To continue with our example, the 2 in EC
2.7.4.4 designates the enzyme as a transferase. The second number, the 7, indicates what group
the enzyme transfers. The third number, the first 4, indicates the destination of the transferred
group. And the last number, the second 4, refines the information given by the third number.

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Table 1
Systematic Classification of Enzymes According to the Enzyme Commission of IUB

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Table 2
Six Basic Class/Types of Enzymes

Table 3
Examples of Enzymes with Trivial Names

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D. ROLE OF COENZYMES AND COFACTORS

Many enzymes carry out their catalytic function relying solely on their protein structure.
Many others require nonprotein components, called cofactors (Table 4). Cofactors may be metal
ions or organic molecules referred to as coenzymes. Cofactors, because they are structurally less
complex than proteins, tend to be stable to heat (incubation in a boiling water bath). Typically,
proteins are denatured under such conditions. Many coenzymes are vitamins or contain vitamins
as part of their structure. Usually coenzymes are actively involved in the catalytic reaction of the
enzyme, often serving as intermediate carriers of functional groups in the conversion of
substrates to products. In most cases, a coenzyme is firmly associated with its enzyme, perhaps
even by covalent bonds, and it is difficult to separate the two. Such tightly bound coenzymes are
referred to as prosthetic groups of the enzyme. The catalytically active complex of protein and
prosthetic group is called the holoenzyme. The protein without the prosthetic group is called the
apoenzyme; it is catalytically inactive.

Table 4
Enzyme Cofactors: Some Metal Ions and Coenzymes and the Enzymes with Which They are
Associated

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E. ENZYME MECHANISM

F. ENZYME KINETICS

Kinetics is the branch of science concerned with the rates of chemical reactions. The
study of enzyme kinetics addresses the biological roles of enzymatic catalysts and how they
accomplish their remarkable feats. In enzyme kinetics, we seek to determine the maximum
reaction velocity that the enzyme can attain and its binding affinities for substrates and
inhibitors. Coupled with studies on the structure and chemistry of the enzyme, analysis of the
enzymatic rate under different reaction conditions yields insights regarding the enzyme’s
mechanism of catalytic action. Such information is essential to an overall understanding of
metabolism.

Significantly, this information can be exploited to control and manipulate the course of
metabolic events. The science of pharmacology relies on such a strategy. Pharmaceuticals, or
drugs, are often special inhibitors specifically targeted at a particular enzyme in order to
overcome infection or to alleviate illness. A detailed knowledge of the enzyme’s kinetics is
indispensable to rational drug design and successful pharmacological intervention.

Table 5
Some Enzymes, their Substrates, and specific Km Values

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G. ENZYME INHIBITION

The action of most enzymes is inhibited by many substances. Inhibition is often specific,
and studies of the relationship between inhibitor structure and activity have been important to the
development of our concepts of active sites and of complementarity of surfaces of biomolecules.
Inhibition of enzymes is also the basis of the action of a very large fraction of important drugs.
Inhibition may be reversible or irreversible, the latter leading to permanent inactivation of the
enzyme. Often, but not always, irreversible inhibition is preceded by reversible binding of the
inhibitor at a complementary site on the enzyme surface.

1. COMPETITIVE INHIBITORS

A competitive inhibitor enters the active site of an enzyme and, thus, prevents the
substrate from entering. This prevention results in a decrease in the number of enzyme-substrate
complexes that form, and, hence, a decrease in the rate of catalysis. In most cases, a portion of
the inhibitor mimics a portion of the substrate. An increase in the substrate concentration
overcomes this inhibition because of the increased probability of a substrate molecule entering
the active site than an inhibitor molecule.Inhibitors with close structural similarities to a substrate
tend to bind to the substrate site. In truly competitive inhibition, substrate and inhibitor not only
compete for the same site but also their binding is reversible and mutually exclusive. The affinity
of the inhibitor for the enzyme is expressed quantitatively through the inhibition constant Ki
which is the dissociation constant of the enzyme inhibitor complex EI : Ki = [E] [I] / [EI]

Competitive inhibition is extremely common and has great significance for metabolic
control and for the effects of drugs and of poisons. Simple ions are often competitive inhibitors.
Since many biochemical substances substances carry negative charges, anions such as Cl–,

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HCO3–, HPO4 2–, and acetate– frequently act as competitive inhibitors. Two special classes of
competitive inhibitors are characterized by slow binding and slow, tight binding to active sites.
Among the very tight-binding inhibitors, which may also be slow to dissociate from active sites,
are transition state inhibitors.

Competitive inhibition occurs if an increase in the concentration of the substrate is

correlated with a decrease in the degree of inhibition. A competitive inhibitor has a similar
chemical structure to the substrate allowing it to bind to the active site of the enzyme. This
means that in competitive inhibition, the enzyme can bind to the substrate or the inhibitor. If the
enzyme binds the substrate, it cannot bind the inhibitor, explaining why an increase in the
concentration of the substrate is associated with a decrease in the degree of inhibition. Also note
that increasing the amount of substrate can overcome the affect of the inhibitor and restore the
increased rate of the catalyzed reaction.

2. NON-COMPETITIVE INHIBITORS

Noncompetitive inhibitors do not enter the active site but instead bind to some other
region of the enzyme. These species usually do not mimic the substrate. This type of inhibitor
reduces the turnover number of the enzyme. Unlike competitive inhibition, an increase in the
substrate does not overcome noncompetitive inhibition. This type of inhibition takes many
different forms, so there is no simple model.

If an inhibitor binds not only to free enzyme but also to the enzyme substrate complex
ES, inhibition is noncompetitive. In this case, S and I do not mutually exclude each other and
both can be bound to the enzyme at the same time. Why does such an inhibitor slow an
enzymatic reaction? In most instances, the structure of the inhibitor does not show a close
similarity to that of substrate, which suggests that the binding of inhibitors is at an allosteric site,
that is, at a site other than that of the substrate. The inhibition of the enzyme may result from a
distortion of the threedimensional structure of the enzyme which is caused by the binding of the
inhibitor. This distortion may be transmitted to the active site even though the inhibitor binds far
from that site. In some cases two distinctly different conformers of the protein may exist, one
binding substrate well and the other binding inhibitor well. In other instances the bound inhibitor
may interfere with the catalytic action by partially overlapping the active site. In either case the
ES complex reacts to give product in a normal way, but the ESI complex reacts more slowly or
not at all.

Binding of a substance to an allosteric site sometimes has the effect of increasing the
activity of an enzyme rather than inhibiting it. This may occur because the activator stabilizes
the conformation that binds substrate best. The quantitative treatment of such activation is
similar to that of inhibition; allosteric inhibitors and activators are often considered together and
are referred to as modifiers or effectors.

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Noncompetitive inhibition occurs if the degree of inhibition is not affected by a change in

the concentration of the substrate. In this case, the inhibitor can bind to the enzyme, or to the
enzyme-substrate complex. The inhibitor does not bind to the same site as the substrate does, so
the substrate can still bind to the enzyme in the presence of the inhibitor. This explains why the
degree of inhibition is not affected by the concentration of the substrate in this case. A
noncompetitive inhibitor affects the catalyzed rate of a reaction.

3. UNCOMPETITIVE INHIBITORS

Uncompetitive inhibition occurs when an increase in the concentration of the substrate

results in an increase in the degree of inhibition. If an inhibitor binds only to ES and not to E,
i.e., K1 = 0, this case is referred to as uncompetitive inhibition. Multisubstrate enzymes with
either ordered sequential or ping-pong mechanisms also often give parallel line plots with
inhibitors.

Uncompetitive inhibitors of liver alcohol dehydrogenase could be used to treat cases of

poisoning by methanol or ethylene glycol. Theaim is to prevent rapid oxidation to the toxic acids
HCOOH and HOCH2COOH, which lower blood pH, while the alcohols are excreted.

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Uncompetitive inhibitors have an advantage over competitive inhibitors as therapeutic agents in

that the inhibition is not overcome when the substrate concentration is saturating.

Enzyme-activated inhibitors (also called suicide substrates, kcat inhibitors, or

mechanism-based inhibitors) are chemically inert until they enter the active site of their target
enzymes. Then, by passing through at least some of the normal stages of the catalytic action of
the enzyme, they are converted to reactive intermediates that can become irreversibly bound to
an enzyme. For example, a halogen atom (F, Cl, Br) together with a proton may be eliminated
from an intermediate to give an unsaturated compound to which a nucleophilic side chain of the
protein adds. Several of these inhibitors are discussed in later chapters. Because of their high
specificity many enzymeactivated inhibitors are potential drugs. One of them, α-
difluoromethylornithine, is said to bind covalently to only one protein in the body of a rat,
namely, ornithine decarboxylase, the target enzyme for the drug.

IMPORTANT

COMPETITIVE INHIBITOR

NONCOMPETITIVE INHIBITOR

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UNCOMPETITIVE INHIBITOR

H. FACTORS AFFECTING THE RATE OF CATALYTIC ACTION

Enzymes can be isolated from cells and their properties studied in a test tube (that is, in
vitro). Different enzymes show different responses to changes in substrate concentration,
temperature, and pH. This section describes factors that influence the reaction velocity of
enzymes. Enzymic responses to these factors give us valuable clues as to how enzymes function
in living cells (that is, in vivo).

1. SUBSTRATE CONCENTRATION

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 Maximal velocity: The rate or velocity of a reaction (v) is the number of substrate
molecules converted to product per unit time. Velocity is usually expressed as μmol of
product formed per minute. The rate of an enzyme-catalyzed reaction increases with
substrate concentration until a maximal velocity (Vmax) is reached. The leveling off of
the reaction rate at high substrate concentrations reflects the saturation with substrate of
all available binding sites on the enzyme molecules present.

 Shape of the enzyme kinetics curve: Most enzymes show Michaelis-Menten kinetics, in
which the plot of initial reaction velocity (vo) against substrate concentration is
hyperbolic. In contrast, allosteric enzymes do not follow Michaelis-Menten kinetics and
show a sigmoidal curve that is similar in shape to the oxygendissociation curve of
hemoglobin.

2. TEMPERATURE

 Velocity increase with temperature: The reaction velocity increases with temperature
until a peak velocity is reached. This increase is the result of the increased number of
substrate molecules having sufficient energy to pass over the energy barrier and form the
products of the reaction.

 Velocity decrease with higher temperature: Further elevation of the temperature causes a
decrease in reaction velocity as a result of temperature-induced denaturation of the
enzyme.

 The optimum temperature for most human enzymes is between 35°C and 40°C. Human
enzymes start to denature (see p. 20) at temperatures above 40°C, but thermophilic
bacteria found in hot springs have optimum temperatures of 70°C.

3. PH
 pH effect on active site ionization: The concentration of protons ([H+]) affects reaction
velocity in several ways. First, the catalytic process usually requires that the enzyme and
substrate have specific chemical groups in either an ionized or unionized state in order to
interact. For example, catalytic activity may require that an amino group of the enzyme
be in the protonated form (−NH3+). Because this group is deprotonated at alkaline pH, the
rate of the reaction declines.

pH effect on enzyme denaturation: Extremes of pH can also lead to

denaturation of the enzyme, because the structure of the catalytically
active protein molecule depends on the ionic character of the amino acid
side chains.

Variable pH optimum: The pH at which maximal enzyme activity is

achieved is different for different enzymes and often reflects the [H+] at

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which the enzyme functions in the body. For example, pepsin, a digestive
enzyme in the stomach, is maximally active at pH 2, whereas other
enzymes, designed to work at neutral pH, are denatured by such an acidic
environment

I. THE REGULATION OF ENZYMATIC ACTIVITY AND METABOLISM

Living cells must operate with controls that provide a stable environment and a relatively
constant supply of materials needed for biosynthesis and for meeting the energy needs of cells.
They must also be responsive to changes in their environment and must be able to undergo
mitosis and reproduce when appropriate. The necessary control of metabolism and of growth is
accomplished largely through mechanisms that regulate the locations, the amounts, and the
catalytic activities of enzymes.

1. Allosteric Effectors in the Regulation of Enzyme Activity (Allosteric Control)

The binding of a substance at an allosteric site with the induction of a conformational

change forms the basis for many aspects of regulation. The term allostery (allosterism) usually
refers to the effects of allosteric modifiers, which may be either inhibitors or activators, on
oligomeric enzymes. However, as we have already seen, monomeric enzymes may also be
subject to allosteric regulation by modifiers. Consider a monomer that contains binding sites for
substrate, inhibitor, and activator and which exists in conformations A and B. Let us assume that
conformer B binds both substrate and activator well but that it binds inhibitor poorly or not at all.
On the other hand, A binds inhibitor well but binds substrate and activator poorly. This simple
combination of two conformers with different binding properties provides a means by which
enzymes can be turned “on” or “off” in response to changing conditions.

If an inhibitory substance builds up to a high concentration within a cell, it binds to

conformer A; if the inhibitor concentration is high enough, virtually all of the enzyme will be
locked in the inactive conformation A. The enzyme will be turned off or at least reduced to a low
activity. On the other hand, in the presence of a high concentration of activator the enzyme will
be turned on because it is locked in the B conformation.

Most intracellular enzymes are oligomeric, and the binding of allosteric effectors leads to
additional interesting effects. Binding constants or dissociation constants must be defined for
both inhibitor and activator to both conformers A and B. Since all species must be taken into
account in the mass balance, the equations are complex.

Noncompetitive inhibition cannot be completely reversed by very high substrate

concentrations.

The significant difference between the two figures is that saturation of the oligomeric
enzyme occurs over a narrower concentration range than does that of the monomer, i.e.,
saturation of the oligomeric enzyme, especially in the presence of inhibitor, is cooperative. Note
that cooperative binding of substrate requires that the free enzyme be largely in conformation T

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College of Education

(A), as it is in the presence of an inhibitor. Allosteric interactions between two identical

molecules, whether of substrate or of effector, are described by Monod et al.80 as homotropic
interactions. Such interactions lead to cooperativity or anticooperativity in binding. Allosteric
interactions between two different molecules, e.g., a substrate and an activator are designated
heterotropic.

2. Multiple Enzyme Forms

Some enzymes have multiple forms known as isozymes or isoenzymes. There are slight
differences in the structures of the forms. These differences lead to differences in the KM and
Vmax values, and, therefore, in the general activity.

3. Covalent Modification

In this form of regulation, the attachment of a group, often a phosphoryl group, alters the
activity of the enzyme. This process is a reversible form of control. Protein kinases catalyze this
type of activation, whereas other enzymes catalyze deactivation.

4. Proteolytic Activation

In this form of regulation, an inactive form of an enzyme — a proenzyme or a zymogen

— often undergoes irreversible conversion to the active form, often through the hydrolysis of
one or more peptide bonds.

EXERCISES:

REFERENCES AND RESOURCES:

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College of Education

APPENDICES:

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