Lesson 4: Basic Concepts of Enzyme Action

Introduction
Enzymes are essential biological catalysts that play a critical role in many chemical reactions in living
organisms. They are responsible for catalyzing a wide range of biochemical reactions, such as digestion,
metabolism, and DNA replication. Understanding the basic concepts of enzyme action is fundamental to
comprehending how these reactions occur and how they are regulated.

In this topic, we will explore the structure and function of enzymes, as well as the mechanisms by which they
catalyze chemical reactions. We will discuss the various factors that affect enzyme activity, including
temperature, pH, substrate concentration, and enzyme concentration. We will also explore the regulation
of enzyme activity and the different types of enzyme inhibitors.

Learning outcomes
By the end of the lesson, you should be able to
1. Define enzymes and describe their role in biological systems.
2. Explain the basic mechanisms by which enzymes catalyze chemical reactions.
3. Discuss the factors that affect enzyme activity.
4. Describe the regulation of enzyme activity and the different types of enzyme inhibitors.

I. Take off

Activity 4.1: Mind Mapping Enzyme Action

Task:
1. Create a mind map or concept map of what you already know about enzymes and their role in
biological systems.
2. Use different colors, shapes, and symbols to represent different ideas and concepts related to
enzymes. Include things like the structure of enzymes, how enzymes work, the different types of
enzymes, and the role of enzymes in biological reactions.
3. Present your created mind map to the rest of the class.

II. Content Focus

Enzymes Are Powerful and Highly Specific Catalysts

Enzymes accelerate the rate of reactions by factors of as much as a million or more (Table 4.1).
Indeed, most reactions in biological systems do not take place at perceptible rates in the absence of
enzymes. Even a reaction as simple as adding water to carbon dioxide is catalyzed by an enzyme—
namely, carbonic anhydrase. This reaction facilitates the transport of carbon dioxide from the tissues
where it is produced to the lungs where it is exhaled. Carbonic anhydrase is one of the fastest known
enzymes. Each enzyme molecule can hydrate 106 molecules of CO2 per second. This catalyzed
reaction is 107 times as fast as the uncatalyzed one. The transfer of CO2 from the tissues to the blood
and then to the lungs would be less complete in the absence of this enzyme.

Table 4.1 Rate enhancement by selected enzymes

Enzyme Nonenzymatic Uncatalyzed rate (kun Catalyzed Rate enhancement

half-life s−1) rate (kcat (kcat s−1/kun
s−1)
s−1)
OMP decarboxylase 78,000,000 2.8 × 10−16 39 1.4 × 1017
years
Staphylococcal nuclease 130,000 years 1.7 × 10−13 95 5.6 × 1014
AMP nucleosidase 69,000 years 1.0 × 10−11 60 6.0 × 1012
Carboxypeptidase A 7.3 years 3.0 × 10−9 578 1.9 × 1011
Ketosteroid isomerase 7 weeks 1.7 × 10−7 66,000 3.9 × 1011
Triose phosphate isomerase 1.9 days 4.3 × 10−6 4,300 1.0 × 109
Chorismate mutase 7.4 hours 2.6 × 10−5 50 1.9 × 106
Carbonic anhydrase 5 seconds 1.3 × 10−1 1 × 106 7.7 × 106
Abbreviations: OMP, orotidine monophosphate; AMP, adenosine monophosphate. Source: Data from A.Radzicka and
R.Wolfenden, Science 267:90–93, 1995.
 Proteolytic Enzymes Illustrate the Range of Enzyme Specificity

Enzymes are highly specific both in the reactions that they catalyze and in their choice of reactants,
which are called substrates. An enzyme usually catalyzes a single chemical reaction or a set of closely
related reactions. Let us consider proteolytic enzymes as an example. The biochemical function of these
enzymes is to catalyze proteolysis, the hydrolysis of a peptide bond:

Proteolytic enzymes differ markedly in their degree of substrate specificity.

Papain, which is found in papaya plants, is quite undiscriminating: it will cleave
any peptide bond with little regard to the identity of the adjacent side chains.
This lack of specificity accounts for its use in meat-tenderizing sauces. Trypsin, a
digestive enzyme, is quite specific and catalyzes the splitting of peptide bonds
only on the carboxyl side of lysine and arginine residues (Figure 4.1A). Thrombin,
an enzyme that participates in blood clotting, is even more specific than trypsin. It
catalyzes the hydrolysis of Arg–Gly bonds in particular peptide sequences only
(Figure 4.1B). The specificity of an enzyme is due to the precise interaction of the
substrate with the enzyme. This precision is a result of the intricate three-
dimensional structure of the enzyme protein.

Figure 4.1 Enzyme specificity. There Are Six Major Classes of Enzymes
(a) trypsin cleaves on the More than a thousand enzymes have been identified—a daunting number for a
carboxyl side of arginine and student of biochemistry. Despite this large number, however, there are only six
lysine residues, whereas (B)
thrombin cleaves arg–Gly bonds
major classes of enzymes, which makes recognizing the function of enzymes much
in particular sequences only. simpler. We will see many members of these classes as we progress in our study
of biochemistry.

1. Oxidoreductases. These enzymes transfer electrons between molecules. In oth- er words, these
enzymes catalyze oxidation–reduction reactions. We will first meet a member of this class,
lactate dehydrogenase, when we consider glycoly- sis, the first pathway in glucose
degradation.
2. Transferases. These enzymes transfer functional groups between molecules. Aminotransferases
are prominent in amino acid synthesis and degradation, where they shuffle amine groups
between donor and acceptor molecules.
3. Hydrolyases. A hydrolyase cleaves molecules by the addition of water. Trypsin, the proteolytic
enzyme already discussed, is a hydrolyase.
4. Lyases. A lyase adds atoms or functional groups to a double bond or removes them to form
double bonds. The lyase fumarase is crucial to aerobic fuel metabolism.
5. Isomerases. These enzymes move functional groups within a molecule. We will meet triose
phosphate isomerase in glycolysis.
6. Ligases. Ligases join two molecules in a reaction powered by ATP hydrolysis. DNA ligase, an
important enzyme in DNA replication, is representative of this class.

The classification of all enzymes into these six classes also allowed the development of a standard
nomenclature for enzymes. Many enzymes have common names that provide little information about
the reactions that they catalyze. Trypsin exemplifies this lack of information. Most other enzymes are
named for their substrates and for the reactions that they catalyze, with the suffix “ase” added. Thus,
a peptide hydrolase is an enzyme that hydrolyzes peptide bonds, whereas ATP synthase is an enzyme
that synthesizes ATP. Common names will be used in this book, but let’s examine the more accurate
nomenclature.

The six groups (classes) of enzymes were subdivided and further subdivided so that a four-digit
number preceded by the letters EC for Enzyme Commission, the entity that developed the classification
system, could precisely identify all enzymes.

Consider trypsin as an example. Trypsin cleaves bonds by the addition of water; consequently, it is a
member of group 3: Hydrolyases. Trypsin cleaves only peptide bonds, and hydrolyases that cleave
peptide bonds are classified as 3.4. Trypsin employs a serine residue to facilitate hydrolysis and

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cleaves the protein chain internally (in contrast with the removal of amino acids from the end of the
polypeptide chain). Such enzymes are placed in sub-sub-group 21 and identified as 3.4.21. Finally,
trypsin cleaves peptide bonds in which the amino acid donating the carboxyl group to the peptide
bond is either lysine or arginine. Thus, the number uniquely identifying trypsin is EC 3.4.21.4. Although
the common names are used routinely, the classification number is used when the precise identity of the
enzyme might be ambiguous.
Many Enzymes Require Cofactors for Activity
Although the chemical repertoire of amino acid functional groups is quite varied (pp. 39–43), they
often cannot meet the chemical needs required for catalysis to take place. Thus, the catalytic activity of
many enzymes depends on the presence of small molecules termed cofactors. The precise role varies
with the cofactor and the enzyme. An enzyme without its cofactor is referred to as an apoenzyme; the
complete, catalytically active enzyme is called a holoenzyme. Cofactors can be subdivided into two
groups: (1) small organic molecules, derived from vitamins, called coenzymes and (2) metals (Table
6.2). Tightly bound coenzymes are called prosthetic (helper) groups. Loosely associated coenzymes are
more like cosub- strates because, like substrates and products, they bind to the enzyme and are
released from it. Coenzymes are distinct from normal substrates not only because they are often
derived from vitamins but also because they are used by a variety of enzymes. Different enzymes that
use the same coenzyme usually carry out similar chemical transformations.

Gibbs Free Energy Is a Useful Thermodynamic Function for Understanding Enzymes

Enzymes speed up the rate of chemical reactions, but the properties of the reaction— whether it can
take place at all—depends on free-energy differences. Gibbs free energy, or more simply free
energy (G), is a thermodynamic property that is a measure of useful energy, or energy that is capable
of doing work. To understand how enzymes, operate, we need to consider only two thermodynamic
properties of the reaction: (1) the free-energy difference (ΔG) between the products and the reactants
and (2) the free energy required to initiate the conversion of reactants into products. The former
determines whether the reaction will take place spontaneously, whereas the latter determines the rate
of the reaction. Enzymes affect only the latter. Let us review some of the principles of thermodynamics
as they apply to enzymes.

The Free-Energy Change Provides Information About the Spontaneity but Not the Rate of a Reaction
The free-energy change of a reaction (ΔG) tells us whether the reaction can take place spontaneously:

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1. A reaction can take place spontaneously only if ΔG is negative. “Spontaneously” in the context of
thermodynamics means that the reaction will take place with- out the input of energy and, in fact,
the reaction releases energy. Such reactions are said to be exergonic.
2. A reaction cannot take place spontaneously if ΔG is positive. An input of free energy is required
to drive such a reaction. These reactions are termed endergonic.
3. In a system at equilibrium, there is no net change in the concentrations of the products and
reactants, and ΔG is zero.
4. The ΔG of a reaction depends only on the free energy of the products (the final state) minus the
free energy of the reactants (the initial state). The ΔG of a reaction is independent of the path (or
molecular mechanism) of the transformation. The mechanism of a reaction has no effect on ΔG. For
example, the ΔG for the transformation of glucose into CO2 and H2O is the same whether it takes
place by combustion or by a series of enzyme-catalyzed steps in a cell.
5. The ΔG provides no information about the rate of a reaction. A negative ΔG indicates that a
reaction can take place spontaneously, but it does not signify whether it will proceed at a
perceptible rate. As will be discussed shortly (p. 103), the rate of a reaction depends on the free
energy of activation (ΔG‡), which is largely unrelated to the ΔG of the reaction.

The Standard Free-Energy Change of a Reaction Is Related to the Equilibrium Constant

As for any reaction, we need to be able to determine ΔG for an enzyme-catalyzed reaction to know
whether the reaction is spontaneous or requires an input of energy. To determine the free-energy
change of the reaction, we need to take into account the nature of both the reactants and the products
as well as their concentrations.
Consider the reaction

The ΔG of this reaction is given by

In which ΔG° is the standard free-energy change, R is the gas constant, T is the absolute temperature,
and [A], [B], [C], and [D] are the molar concentrations of the reactants. ΔG° is the free-energy change
for this reaction under standard conditions—that is, when each of the reactants A, B, C, and D is
present at a concentration of 1.0 M (for a gas, the standard state is usually chosen to be 1
atmosphere) before the initiation of the reaction, and the temperature is 298 K (298 kelvins, or 25°C).
Thus, the ΔG of a reaction depends on the nature of the reactants (expressed in the ΔG° term of
equation 1) and on their concentrations (expressed in the logarithmic term of equation 1).
A convention has been adopted to simplify free-energy calculations for biochemical reactions. The
standard state is defined as having a pH of 7. Consequently, when H+ is a reactant, its concentration
has the value 1 (correspond- ing to a pH of 7) in the numbered equations that follow. The
concentration of water also is taken to be 1 in these equations. The standard free-energy change at
pH 7, denoted by the symbol ΔG°′, will be used throughout this book. The kilojoule (kJ) and the
kilocalorie (kcal) will be used as the units of energy. As stated in Chapter 2, 1 kJ is equivalent to
0.239 kcal.
A simple way to determine the ΔG°′ is to measure the concentrations of reactants and products
when the reaction has reached equilibrium. At equilibrium, there is no net change in the concentrations
of reactants and products; in essence, the reaction has stopped and ΔG = 0. At equilibrium, equation
1 then becomes

and also

The equilibrium constant under standard conditions, K′eq, is defined as

Substituting equation 4 into equation 3 gives

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which can be rearranged to give

It is important to stress that whether the ΔG for a reaction is larger, smaller, or the same as ΔG°′
depends on the concentrations of the reactants and products. The criterion of spontaneity for a
reaction is ΔG, not ΔG°′. This point is important because reactions that are not spontaneous, on the
basis of ΔG°′, can be made spontaneous by adjusting the concentrations of reactants and products. This
principle is the basis of the coupling of reactions to form metabolic pathways (Chapter 15).

Enzymes Alter the Reaction Rate but Not the Reaction

Equilibrium
Because enzymes are such superb catalysts, it is tempting
to ascribe to them powers that they do not have. An
enzyme cannot alter the laws of thermodynamics and
consequently cannot alter the equilibrium of a chemical
reaction. Consider an enzyme-catalyzed reaction, the
conversion of substrate, S, into product, P. Figure 6.2
graphs the rate of product formation with time in the
presence and absence of enzyme. Note that the amount
of product formed is the same whether or not the enzyme
is present, but, in the present example, the amount of
Figure 6.2 Enzymes accelerate the reaction product formed in seconds when the enzyme is present
rate. the same equilibrium point is reached but might take hours or centuries to form if the enzyme were
much more quickly in the presence of an absent (Table 6.1).
enzyme.

Enzymes Facilitate the Formation of the Transition State

The free-energy difference between reactants and products accounts for the equilibrium of a
reaction, but enzymes accelerate how quickly this equilibrium is attained. How can we explain the rate
enhancement in terms of thermodynamics? To do so, we have to consider not the end points of the
reaction but the chemical pathway between the end points.

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 A chemical reaction of substrate S to form product P goes through a transition state X‡ that has a
higher free energy than does either S or P. The double dagger denotes the transition state. The
transition state is a fleeting molecular structure that is no longer the substrate but is not yet the product.
The transition state is the least-stable and most-seldom-occurring species along the reaction pathway
because it is the one with the highest free energy.

The difference in free energy between the transition state and the substrate is called the free energy
of activation or simply the activation energy, symbolized by ΔG‡ (Figure 6.3):

Note that the energy of activation, or ΔG‡, does not enter into the final ΔG calculation for the
reaction, because the energy that had to be added to reach the transition state is released when the
transition state becomes the product. The activation energy immediately suggests how enzymes
accelerate the reaction rate without altering ΔG of the reaction: enzymes function to lower the
activation energy. In other words, enzymes facilitate the formation of the transition state.

The combination of substrate and enzyme creates a

reaction pathway whose transition-state energy is lower
than what it would be without the enzyme (Figure 6.3).
Because the activation energy is lower, more molecules
have the energy required to reach the transition state
and more product will be formed faster. Decreasing the
activation barrier is analogous to lowering the height of
a high-jump bar; more athletes will be able to clear the
bar. The essence of catalysis is stabilization of the
transition state

Figure 6.3 Enzymes decrease the activation

energy. enzymes accelerate reactions by
decreasing ΔG‡, the free energyof activation.

The Formation of an Enzyme–Substrate Complex Is the First Step in Enzymatic Catalysis

Much of the catalytic power of enzymes comes from their binding to and then altering the structure of
the substrate to promote the formation of the transition state. Thus, the first step in catalysis is the
formation of an enzyme–substrate (ES) complex. Substrates bind to a specific region of the enzyme
called the active site. Most enzymes are highly selective in the substrates that they bind. Indeed, the
catalytic specificity of enzymes depends in part on the specificity of binding.

The Active Sites of Enzymes Have Some Common Features

The active site of an enzyme is the region that binds the substrates (and the cofactor, if any). It also
contains the amino acid residues that directly participate in the making and breaking of bonds. These
residues are called the catalytic groups. 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 ΔG‡ of the reaction, thus providing the rate-enhancement characteristic of
enzyme action. Recall from Chapter 4 that proteins are not rigid structures, but are flexible and exist in
an array of conformations. Thus, the interaction of the enzyme and substrate at the active site and the
formation of the transition state is a dynamic process. Although enzymes differ widely in structure,
specificity, and mode of catalysis, a number of generalizations concerning their active sites can be
stated:

1. The active site is a three-dimensional cleft or

crevice formed by groups that come from different parts
of the amino acid sequence: indeed, amino acids near to
one another in the primary structure are often sterically
constrained from adopting the structural relations
necessary to form the active site. In lysozyme, the
important groups in the active site are contributed by
residues numbered 35, 52, 62, 63, 101, and 108 in the
sequence of 129 amino acids (Figure 6.4). Lysozyme,
found in a variety of organisms and tissues including
human tears, degrades the cell walls of some bacteria.

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 2. The active site takes up a small part of the total
volume of an enzyme. Although most of the amino acid
residues in an enzyme are not in contact with the
Figure 6.4 Active sites may include distant substrate, the cooperative motions of the enzyme as a
residues. (a) ribbon diagram of the enzyme whole help to correctly position the catalytic residues at
lysozyme with several components of the active site
shown in color. (B) a schematic representation of
the active site. Experimental attempts to reduce the size
the primary structure of lysozyme shows that the of a catalytically active site. Experimental attempts to
active site is composed of residues that come from reduce the size of a catalytically active enzyme show that
different parts of the polypeptide chain. [Drawn the minimum size requires about 100 amino acid residues.
from 6LYZ.pdb.] In fact, nearly all enzymes are made up of more than 100
amino acid residues, which gives them a mass greater
than 10 kDa and a diameter of more than 25 Å,
suggesting that all amino acids in the protein, not just
those at the active site, are ultimately required to form a
functional enzyme.

3. Active sites are unique microenvironments. The close association between the active site and the
substrate means that water is usually excluded from the active site unless it is a reactant. The nonpolar
microenvironment of the cleft enhances the binding of substrates as well as catalysis. Nevertheless, the cleft
may also contain polar residues, some of which may acquire special properties essential for substrate
binding or catalysis. The internal positions of these polar residues are biologically crucial exceptions to the
general rule that polar residues are located on the surface of proteins, exposed to water.

4. Substrates are bound to enzymes by multiple weak attractions. The noncovalent interactions
between the enzyme and the substrate in ES complexes are much weaker than covalent bonds. These weak
reversible interactions are mediated by electrostatic interactions, hydrogen bonds, and van der Waals
forces, powered by the hydrophobic effect. Van der Waals forces become significant in binding only when
numerous substrate atoms simultaneously come close to many enzyme atoms. Hence, to bind as strongly as
possible, the enzyme and substrate should have complementary shapes.

Figure 6.5 Lock-and-key model of

enzyme–substrate binding. In this model,
the active site of the unbound enzyme is
complementary in shape to the substrate.

5. The specificity of binding depends on the precisely defined arrangement of atoms in an active
site. Because the enzyme and the substrate interact by means of short-range forces that require close
contact, a substrate must have a matching shape to fit into the site. Emil Fischer’s analogy of the lock and
key (Figure 6.5), expressed in 1890, has proved to be highly stimulating and fruitful. However, we now
know that enzymes are flexible and that the shapes of the active sites can be markedly modified by the
binding of substrate, a process of dynamic recognition called induced fit (Figure 6.6). Moreover, the
substrate may bind to only certain conformations of the enzyme, in what is called conformation selec- tion.
Thus, the mechanism of catalysis is dynamic, involving structural changes with multiple intermediates of both
reactants and the enzyme.

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 Figure 6.6 Induced-fit model of
enzyme–substrate binding. In this model,
the enzyme changes shape on substrate
binding. the active site forms a shape
complementary to the substrate only after the
substrate has been bound.

The Binding Energy Between Enzyme and Substrate Is Important for Catalysis
Enzymes lower the activation energy, but where does the energy to lower the activation energy come
from? Free energy is released by the formation of a large number of weak interactions between a
complementary enzyme and substrate. The free energy released on binding is called the binding energy.
Only the correct substrate can participate in most or all of the interactions with the enzyme and thus
maximize binding energy, accounting for the exquisite substrate specificity exhibited by many enzymes.
Furthermore, the full complement of such interactions is formed only when the substrate is in the transition
state. Thus, the maximal bind- ing energy is released when the enzyme facilitates the formation of the
transition state. The energy released by the interactions between the enzyme and the sub- strate can be
thought of as lowering the activation energy. The interaction of the enzyme with the substrate and reaction
intermediates is fleeting, with molecular

Figure 6.7 Inhibition by transition-state analogs. (a) the isomerization of l-proline tod-proline
by proline racemase, a bacterial enzyme, proceeds through a planar transition state in which the α-
carbon atom is trigonal rather than tetrahedral. (B) pyrrole 2-carboxylate, a transition-state analog
because of its trigonal geometry, is a potent inhibitor of proline racemase.

movements resulting in the optimal alignment of functional groups at the active site so that maximum
binding energy occurs only between the enzyme and the transition state, the least-stable reaction
intermediate. However, the transition state is too unstable to exist for long. It collapses to either substrate
or product, but which of the two accumulates is determined only by the energy difference between the
substrate and the product—that is, by the ΔG of the reaction.

Transition-State Analogs Are Potent Inhibitors of Enzymes

The importance of the formation of the transition state to enzyme catalysis is demonstrated by the study of
compounds that resemble the transition state of a reaction but are not capable of being acted on by the
enzyme. These mimics are called transition-state analogs. The inhibition of the enzyme proline racemase is
an instructive example. The racemization of proline proceeds through a transition state in which the
tetrahedral α-carbon atom has become trigonal (Figure 6.7). This picture is supported by the finding that
the inhibitor pyrrole 2-carboxylate binds to the racemase 160 times as tightly as does proline. The α-
carbon atom of this inhibitor, like that of the transition state, is trigonal . An analog that also carries a
negative charge on α-carbon would be expected to bind even more tightly. In general, highly potent and
specific inhibitors of enzymes can be produced by synthesizing compounds that more closely resemble the
transition state than the substrate itself. The inhibitory power of transition-state analogs underscores the
essence of catalysis: selective binding of the transition state.
If our understanding of the importance of the transition state to catalysis is correct, then antibodies that
recognize transition states should function as catalysts. Antibodies have been generated that recognize the
transition states of certain reactions, and these antibodies, called catalytic antibodies or abzymes, do
indeed function as enzymes.

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III. Self-check

Complete the following questions:

1. Enzymes accelerate reactions by factors of ___________ or more.
2. What is the enzyme that catalyzes the reaction of adding water to carbon dioxide?
_____________
3. What is the role of carbonic anhydrase in the body? _____________________________
4. What is the biochemical function of proteolytic enzymes? ________________________
5. What is the substrate of proteolytic enzymes called? ___________________________
6. What is the name of the proteolytic enzyme found in papaya plants? _______________
7. What is the role of trypsin in the body? ______________________________________
8. What type of peptide bonds does thrombin hydrolyze? ___________________________
9. What is the precise interaction that determines enzyme specificity? _______________
10. How many major classes of enzymes are there? ________________________________
11. The first step in enzymatic catalysis is the formation of an enzyme-substrate (ES)
_______________.
12. The active site of an enzyme contains amino acid residues that directly participate in the making
and breaking of _______________.
13. The active site is a three-dimensional cleft or crevice formed by groups that come from different
parts of the _______________ sequence.
14. The minimum size required for a catalytically active enzyme is about _______________ amino
acid residues.
15. The nonpolar microenvironment of the cleft enhances the binding of substrates as well as
_______________.
16. Substrates are bound to enzymes by multiple _______________ interactions.
17. The specificity of binding depends on the precisely defined arrangement of _______________ in
an active site.
18. Enzymes lower the activation energy by the formation of a large number of weak interactions
between a complementary enzyme and _______________.
19. The free energy released on binding is called the _______________ energy.
20. The full complement of interactions between enzyme and substrate is formed only when the
substrate is in the _______________ state.

IV. Self-Reflect

Task: Research a specific enzyme and create a presentation explaining its function, substrate, and
specificity. Moreover. explain which of the six major classes of enzymes the enzyme belongs to and
how its name is determined according to the Enzyme Commission's nomenclature system.

V. References

1. Ferrier, D. R. Lippincott Illustrated Reviews: Biochemistry, 7th Ed. Philadelphia: Lippincott Williams
& Wilkins, 2017.
2. Nelson, D. L., & Cox, M. M. Lehninger principles of biochemistry, 6th Ed. New York: W. H. Freeman
& Company, 2013.
Tymoczko, J. L., Berg, J. M., & Stryer, L. Biochemistry: A short course, 3rd Ed. New York: W. H. Freeman &
Company, 2015