Lesson 6.

3: Enzymes

Lesson Summary
Most of the proteins found in the body functions as enzymes. Enzymes are biological molecules
that speed up a reaction. A reaction inside the cell could take years without the aid of an
enzyme.

Learning Outcomes
At the end of this module, students will be able to
1. Tell what enzymes can and cannot do
2. Describe how enzymes speeds up a reaction
3. Differentiate competitive and non-competitive inhibition

Motivations Question
What are enzymes? What would happen if there are no enzymes in the body? How do enzymes
work?

Discussion
In the preceding lessons, we saw that proteins play crucial roles in nearly all biological
processes in catalysis, signal transmission, and structural support. This remarkable range of
functions arises from the existence of thousands of
proteins, each folded into a distinctive 3D structure that enables it to interact with one or more
of a highly diverse array of molecules. A major goal of biochemistry is to determine how amino
acid sequences specify the conformations of proteins. Other goals are to learn how individual
proteins bind specific substrates and other molecules, mediate catalysis, and transduce
energy and information. Hence, the purification of the protein of interest is the indispensable
first step in a series of studies aimed at exploring protein function.
Proteins can be separated from one another on the basis of solubility, size, charge, and binding
ability. When a protein has been purified, the amino acid sequence can be determined. The
strategy is to divide and conquer, to obtain specific fragments that can be readily sequenced.
Automated peptide sequencing and the application of recombinant DNA methods are providing
a wealth of amino acid sequence data that are opening new vistas. To understand the
physiological context of a protein, antibodies are choice probes for locating proteins in vivo and
measuring their quantities. Monoclonal antibodies able to probe for specific proteins can be
obtained in large amounts. The synthesis of peptides is possible, which makes feasible the
synthesis of new drugs, functional protein fragments, and antigens for inducing the formation of
specific antibodies.
Listed below are the different biological functions of proteins in living systems:
1. The major function of food proteins is to provide the major organic structures of the
protoplasmic machine, although excess is utilized as a source of energy.
2. The chemical processes involved in the digestion of foods and also in the utilization or
metabolism of foods in tissues are, in general, catalyzed by substances known as
enzymes—which are proteins. The enzymes represent the largest class of proteins (so
they will be discussed further in this module). Nearly 2000 different kinds of enzymes
are known, each catalyzing a different kind of chemical reaction. Enzymes are highly
specific in function.
An example is cytochrome C which transfers electrons toward molecular oxygen during
respiration. Also, DNA polymerases and amino acid-activating enzymes participate in the
biosynthesis of cell components. Each type of enzyme contains an active site to which
its specific substrate is bound during the catalytic cycle. Many enzymes contain a single
polypeptide chain (no quaternary structure) while contain two or more. Some enzymes
called regulatory or allosteric enzymes are further specialized to serve a regulatory
function in addition to their catalytic activity. Virtually, all enzymes are globular proteins.
3. Another class of proteins have the function of storing amino acids and using them as
building blocks for the growing embryo (e.g., ovalbumin of egg white, casein of milk, and
gleadin of wheat).
4. Some proteins have a transport function; they are capable of binding and transporting
specific types of molecules via the blood.
Example include serum albumin which binds free fatty acids tightly and thus serves to
transport these molecules between adipose (fatty) tissue and other tissues or organs in
vertebrates.
The lipoprotein of blood plasma transports lipids between the intestine, liver and
adipose (fatty) tissues. Hemoglobin of vertebrate erythrocytes transports oxygen from
the lungs to the tissues.
Invertebrates have other types of oxygen-carrying protein and molecules such as
hemocyanine.
5. Some proteins also serve as essential elements in contractive and motile systems. Actin
and myosin are the two major protein elements of the contractile system of skeletal
muscle. In muscles, these proteins are arranged in parallel arrays and slide along each
other during contraction.
6. Some proteins have a protective or defensive function. The blood protein thrombin and
fibrinogen participate in blood clotting and thus prevent the loss of blood from the
vascular system of vertebrates, but the most important protective proteins are
antibodies or immuno-globulins which combine with and neutralize foreign proteins and
other substances that happen to gain entrance into the blood tissues of a given
vertebrate.
7. Some proteins are extremely toxic to higher animals in very small amounts. These are
called toxins. They include ricin of the castor bean, gossypol of the cotton seed and
hemagglutinins of legumes and certain diseases of both plants and animals are caused
 by substances called viruses. A few of these have been isolated and purified and have
been identified as very complex proteins.
8. Some proteins also function as hormones. Hormones are chemical substances
produced in one part of plant or animal’s body by ductless (endocrine) glands and
produce its effect on distant parts of the body. Much of the integration and regulation of
physiological processes in the body is accomplished through these hormones.
Examples include: somatotropin (growth hormone) – a hormone of anterior pituitary
gland. Insulin – secreted by certain specialized cells of the pancreas. It regulates
glucose metabolism and its deficiency in man causes a disease called diabetes mellitus.
9. Another class of proteins serves as structural elements. In vertebrates, the fibrous
protein, collagen, is the major extracellular structural protein in connective tissues and
bone. Collagen-fibrils, by forming a structural continuum also help to bind a group of
cells together to form a tissue.
The other fibrous proteins in vertebrates are elastin of yellow elastic tissue and α-keratin
present in skin, feathers, nails, hoofs. Cartilage contains not only collagen but also
glycoproteins, which enclose mucous secretions and synovial fluid in the joints of
vertebrates with a slippery, lubricating quality.
10. Besides these major classes of protein, others have unusual functions. Spiders and
silkworms secrete a thick solution of the protein fibroin, which quickly solidifies into an
insoluble thread of exceptional tensile strength used to form webs or cocoons. The
blood of some fishes living in subzero Antarctic waters contains a protein that keeps the
blood from freezing. This protein is called “antifreeze protein”. Monellin is a sweet-
tasting protein found in some fruits. It does not taste sweet when denatured. Unlike
sugars it tastes sweet but does not cause fattening. It should be noted that all proteins,
including those having intense biological or toxic effects, are built from the same 20
amino acids which by themselves have relatively little biological activity or toxicity.

Enzymes: Basic Concepts and Kinetics

As you have learned from the previous lesson, the various properties of amino acids that make
up the protein, and the assembly of the subunits from primary to tertiary structure allows them
to have various several functions. One of these are the enzymes.
Enzymes, the catalysts of biological systems, are remarkable molecular devices that determine
the patterns of chemical transformations. They also mediate the transformation of one form of
energy into another. The most striking characteristics of enzymes are their catalytic power and
specificity. Catalysis takes place at a particular site on the enzyme called the active site. Nearly
all known enzymes are proteins. However, proteins do not have an absolute monopoly on
catalysis; the discovery of catalytically active RNA molecules provides compelling evidence that
RNA was an early biocatalyst.
Proteins are highly effective catalysts for an enormous diversity of chemical reactions because
of their capacity to specifically bind a very wide range of molecules. Remember that the
structure of proteins are formed due to the intermolecular forces. By utilizing the full repertoire
of intermolecular forces, enzymes bring substrates or reactants together in an optimal
orientation, the prelude to making and breaking chemical bonds. And then, they catalyze
reactions by stabilizing transition states, the highest-energy species in reaction pathways. By
selectively stabilizing a transition state, an enzyme determines which one of several potential
chemical reactions actually takes place. Take a look at Figure 19A. The y-axis is the Free
Energy. This pertains to the energy of the substance (e.g., reactant, product, transition state).
Initially, we have the reactants having a specific magnitude of energy. As the reaction
progresses, the energy of the reactants will rise due to the formation of a transition state (TS). A
TS is an unstable molecule that looks like both the reactant and the product. In order to reach the
TS, the “free energy of activation” or the “activation energy” must be overcome. This is like a
mountaineer climbing to the top of the mountain.
Now, because the TS looks both like the reactant and the product, it can choose to form the
“products” or it can choose to go back to the “reactants”. Now, why would the TS choose to
form into either product or reactant? Simple. Because the TS is unstable; hence, it would
choose to convert itself into a more stable molecule. A more stable molecule is that which has
a lower free energy. In this case, both the products and reactants have low free energy.
So what directs the TS to form products or to go back to the reactants? This is where we
introduce “thermodynamic versus kinetic” control.
Thermodynamic control means that the final energy of the system (in this case, Efinal = energy of
the products) will be lower than that of the initial energy of the system (Einitial = energy of the
reactants). From this, we could say that the reaction in Figure 19A is “thermodynamically
favorable”.
So, if a reaction is “spontaneous”, does it mean that it will be fast?
Let us consider the reaction of glucose and oxygen gas to produce carbon dioxide and water.
This reaction requires a number of enzymatic catalysts:
Glucose + 6O2 → 6CO2 + 6H2O
This reaction is thermodynamically favorable (spontaneous in the thermodynamic sense)
because its free energy change is negative (ΔG°= -2880 kJ/mol).
Note that the term spontaneous does not mean “instantaneous.” As you see in Figure 19, the
activation energy affects the rate of the reaction. So what the catalyst does is that it lowers the
activation energy of the reaction, thereby increasing the reaction rate.
Figure 1. Free energy profile (Campbell et al.)

Will a reaction go faster If you raise the temperature?

Raising the temperature of a reaction mixture increases the energy available to the reactants to
reach the transition state. Consequently, the rate of a chemical reaction increases with
temperature. One might be tempted to assume that this is universally true for biochemical
reactions. In fact, increase of reaction rate with temperature occurs only to a limited extent with
biochemical reactions. It is helpful to raise the temperature at first, but eventually there comes a
point at which heat denaturation of the enzyme is reached.
Many enzymes require cofactors for activity

The catalytic activity of many enzymes depends on the presence of small molecules termed
cofactors, although the precise role varies with the cofactor and the enzyme. Such an enzyme
without its cofactor is referred to as an apoenzyme; the complete, catalytically active enzyme is
called a holoenzyme.

Apoenzyme + Cofactor = Holoenzyme

Cofactors can be subdivided into two groups: metals and small organic molecules (Table 2).
The enzyme carbonic anhydrase, for example, requires Zn2+ for its activity. Glycogen
phosphorylase, which mobilizes glycogen for energy, requires the small organic molecule
pyridoxal phosphate (PLP). Cofactors that are small organic molecules are called coenzymes.
Often derived from vitamins, coenzymes can be either tightly or loosely bound to the enzyme. If
tightly bound, they are called prosthetic groups. Loosely associated coenzymes are more like
co-substrates because they bind to and are released from the enzyme just as substrates and
products are. The use of the same coenzyme by a variety of enzymes and their source in
vitamins sets coenzymes apart from normal substrates, however. Enzymes that use the same
coenzyme are usually mechanistically similar.
Figure 2. The activity of an enzyme is responsible for the glow of the luminescent jellyfish. The enzyme aequorin
catalyzes the oxidation of a compound by oxygen in the presence of calcium to release CO2 and light (Stryer et al.)

Figure 3. The effect of Temperature on enzyme activity (Campbell et al.)

Table 1. Enzymes and cofactors (Styer at al.)

COFACTOR ENZYME
Coenzyme
Thiamine pyrophosphate Pyruvate
dehydrogenase
Flavin adenine nucleotide Monoamine oxidase
Nicotinamide adenine Lactate dehydrogenase
dinucleotide
Pyridoxal phosphate Glycogen
phosphorylase
Coenzyme A (CoA) Acetyl CoA carboxylase
Biotin Pyruvate carboxylase
5' -Deoxyadenosyl cobalamin Methylmalonyl mutase
Tetrahydrofolate Thymidylate synthase
Metal
Zn2+ Carbonic anhydrase
Zn2+ Carboxypeptidase
Mg2+ EcoRV
Mg2+ Hexokinase
Ni2+ Urease
Mo Nitrate reductase
Se Glutathione peroxidase
Mn2+ Superoxide dismutase
K+ Propionyl CoA
carboxylase

Enzymes are Classified on the Basis of the type of Reactions that they Catalyze

Many enzymes have common names that provide little information about the reactions that
they catalyze. For example, a proteolytic enzyme secreted by the pancreas is called trypsin.
Most other enzymes are named for their substrates and for the reactions that they catalyze,
with the suffix "ase" added. Thus, an ATPase is an enzyme that breaks down ATP, whereas ATP
synthase is an enzyme that synthesizes ATP. To bring some consistency to the classification of
enzymes, in 1964 the International Union of Biochemistry established an Enzyme Commission
to develop a nomenclature for enzymes. Reactions were divided into six major groups
numbered 1 through 6 (Table 3). These groups were subdivided and further subdivided, so that
a four-digit number preceded by the letters EC for Enzyme Commission could precisely identify
all enzymes. Consider as an example nucleoside monophosphate (NMP) kinase. It catalyzes the
following reaction:
 ATP + NMP → ADP + NDP
NMP kinase transfers a phosphoryl group from ATP to NMP to form a nucleoside diphosphate
(NDP) and ADP. Consequently, it is a transferase, or member of group 2. Many groups in
addition to phosphoryl groups, such as sugars and carbon units, can be transferred.
Transferases that shift a phosphoryl group are designated 2.7. Various functional groups can
accept the phosphoryl group. If a phosphate is the acceptor, the transferase is designated 2.7.4.
The final number designates the acceptor more precisely. In regard to NMP kinase, a nucleoside
monophosphate is the acceptor, and the enzyme's designation is EC 2.7.4.4. Although the
common names are used routinely, the classification number is used when the precise identity
of the enzyme might be ambiguous.

Table 2. Classes of enzymes (Stryer et al.)

Class Type of reaction Example

1.
Oxidation-reduction Lactate dehydrogenase
Oxidoreductases
Nucleoside
2. Transferases Group transfer monophosphate kinase
(NMP kinase)
Hydrolysis reactions
3. Hydrolases (transfer of functional Chymotrypsin
groups to water)
Addition or removal of
4. Lyases groups to form double Fumarase
bonds
Isomerization
Triose phosphate
5. Isomerases (intramolecular group
isomerase
transfer)
Ligation of two substrates
Aminoacyl-tRNA
6. Ligases at the expense of ATP
synthetase
hydrolysis

Enzyme-Substrate Binding

In an enzyme-catalyzed reaction, the enzyme binds to the substrate (one of the reactants) to
form a complex. The formation of the complex leads to the formation of the transition-state
species, which then forms the product. The nature of transition states in enzymatic reactions is
a large field of research in itself, but some general statements can be made on the subject. A
substrate binds, usually by noncovalent interactions, to a small portion of the enzyme called the
active site, frequently situated in a cleft or crevice in the protein and consisting of certain amino
acids that are essential for enzymatic activity (Figure 16). The catalyzed reaction takes place at
the active site, usually in several steps.
Figure 4. Two models for the binding of substrate and enzyme (Campbell et al.)

Why do enzymes bind to the substrate?

As shown in Figure 16, there are two models of enzyme-substrate binding. First is the lock-and-
key model. In this model, there is huge similarity in the shape and geometry of the substrate’s
active site and the enzyme’s binding site which allows perfect binding. In the induced-fit model,
on the other hand, takes into account the flexibility of the conformation of protein structures.
According to this induced-fit model, the binding of the substrate induces a conformational
change in the enzyme that results in a complementary fit after the substrate is bound.

Michaelis-Menten Enzyme Kinetics

In a mathematical description of enzyme action developed by Leonor Michaelis and Maud

Menten in 1913, two constants, Vmax and Km, play an important role. These constants are
important to know, both to understand enzyme activity on the macroscale and to understand
the effects of different types of enzyme inhibitors.
Maximal Velocity (Vmax): Increasing the substrate concentration indefinitely does not increase
the rate of an enzyme-catalyzed reaction beyond a certain point. This point is reached when
there are enough substrate molecules to completely fill (saturate) the enzyme's active sites. The
maximal velocity, or Vmax, is the rate of the reaction under these conditions. Vmax reflects how
fast the enzyme can catalyze the reaction. In the figure below, Vmax is given by the asymptote to
the velocity curve as the substrate concentration is extrapolated to infinity. Notice that K m stays
constant for the two enzymes described here.
Michaelis Constant (Km): Enzymes have varying tendencies to bind their substrates (affinities).
An enzyme's Km describes the substrate concentration at which half the enzyme's active sites
are occupied by substrate. A high Km means a lot of substrate must be present to saturate the
enzyme, meaning the enzyme has low affinity for the substrate. On the other hand, a low
Km means only a small amount of substrate is needed to saturate the enzyme, indicating a high
affinity for substrate. Graphically, the Km is the substrate concentration that gives the enzyme
one-half of its Vmax. Although it may look like the Vmax drops, if the graph is extended along the x-
axis, the Vmax stays constant for the two enzymes described here.
Figure 5. Michaelis-Menten Kinetics [6].

Types of Enzyme Inhibition

Sometimes enzymes need to be turned off. For example, a complicated system of enzymes and
cells in your blood has the task of forming a clot whenever you are cut, to prevent death from
blood loss. If these cells and enzymes were active all the time, your blood would clot with no
provocation and it would be unable to deliver oxygen and nutrients to the peripheral tissues in
your body. So enzymes have evolved mechanisms to be turned off, which usually
involve inhibitors, molecules that bind to an enzyme and prevent it from catalyzing its reaction.
There are three general kinds of inhibitors: competitive, noncompetitive, and mixed inhibitors.

COMPETITIVE INHIBITION

In competitive inhibition, a molecule similar to the substrate but unable to be acted on by the
enzyme competes with the substrate for the active site. Because of the presence of the
inhibitor, fewer active sites are available to act on the substrate. But since the enzyme's overall
structure is unaffected by the inhibitor, it is still able to catalyze the reaction on substrate
molecules that do bind to an active site. Note that since the inhibitor and substrate bind at the
same site, competitive inhibition can be overcome simply by raising the substrate
concentration.

At the macroscopic level, the effect of competitive inhibition is to increase the substrate
concentration required to achieve a given reaction velocity; in other words, to raise the Km. The
Vmax is unchanged, however.

NON-COMPETITIVE INHIBITION
The other type of inhibition is noncompetitive inhibition. In noncompetitive inhibition, a molecule
binds to an enzyme somewhere other than the active site. This changes the enzyme's three-
dimensional structure so that its active site can still bind substrate with the usual affinity, but is
no longer in the optimal arrangement to stabilize the transition state and catalyze the reaction.

On the macroscopic scale, noncompetitive inhibition lowers the Vmax. Thus, the enzyme simply
cannot catalyze the reaction with the same efficiency as the uninhibited enzyme. Note that
noncompetitive inhibition cannot be overcome by raising the substrate concentration like
competitive inhibition can.

Learning Tasks/Activities
1. Read on the passage below and elaborate your learning. What have you concluded?
Think about how you can make use of enzymes in health, nature, medicine and other
discipline.
2. Reflect: If only a few of the amino acid residues of an enzyme are involved in its catalytic
activity, why does the enzyme need such a large number of amino acids?

Assessment
General Instructions: Write your name on every page of the answer sheet.
1. What are enzymes?
2. List 20 hydrolytic products of protein.
3. Describe the two models of enzyme-substrate binding.
4. Describe the significance of Km and Vmax.
5. Differentiate competitive versus non-competitive inhibitor.
6. Would you expect glucose to be an inhibitor of fructose? Explain your reasoning.
7. Would you expect sucrose to be an inhibitor of glucose? If yes, will it be competitive or
non-competitive? Explain.
8. Differentiate active site from an allosteric site in 1-2 sentences.
9. Distinguish between the lock-and-key model and the induced-fit model in not more than
3 sentences.