Enzyme

s
ENZYMES
Definition
Enzymes are biocatalysts mainly proteins in nature that regulate the rate of all
biochemical reactions.

Common Features
- All are produced by living cells and can act outside these cells.
- They are needed in very small amounts.
- They accelerate the reaction without affecting its equilibrium (decrease energy of
activation).
- They are not changed chemically by the end of the reaction.
- They are highly specific (act on a specific substrate or inter-related substrates).

Nomenclature
- Most commonly used enzyme names have the suffix “-ase” attached to the substrate
of the reaction (for example, glucosidase, urease, sucrase), or to a description of the
action performed (for example, lactate dehydrogenase and adenylyl cyclase).
- Some enzymes retain their original trivial names, which give no hint of the
associated enzymatic reaction, for example, trypsin and pepsin.

Enzyme Specificity
- Enzymes are highly specific in their action, interacting with one or a few specific
substrates and catalyzing only one type of chemical reactions.
- The specificity of enzymes is due to the nature and arrangement of the chemical
groups at the catalytic site (see latter). This allows the enzyme to unite and activate
only one substrate or a small number of structurally related substrates.
Importance of enzyme specificity: -
The relatively low specificity of the digestive enzymes allows only a few enzymes to
digest all foodstuffs. The high specificity of intracellular enzymes allows metabolic
pathways to work and to be regulated more properly.

Chemical Nature
- Except for ribozymes, virtually all enzymes are protein in nature; some are simple
proteins while others are conjugated proteins (holoenzyme).
- Holoenzyme refers to the active enzyme with its nonprotein component (cofactor),
whereas the enzyme without its cofactor is termed an apoenzyme and is inactive.
- Cofactors are organic (Coenzymes) or inorganic molecules (Metal ions) that are
required for the activity of a certain conjugated enzymes. They participate in
substrate binding or catalysis.

Mechanism of Enzyme Action (induced fit model)

The Active Site:
- Enzyme molecules contain a special pocket or cleft called the active site.
- The active site contains amino acid side chains that create a three-dimensional
surface complementary to the substrate.
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- s active site binds the substrate, forming an enzyme–substrate (ES) complex;
The
ES complex is converted to an enzyme–product (EP) complex that subsequently
dissociates to enzyme and product.
- The induced fit model states that when substrates bind to an enzyme, they induce a
conformational change analogous to placing a hand (substrate) into a glove
(enzyme).
T1
Role of Enzymes in Catalysis
A- Lower activation energy Activation energy of
Uncatalyzed reaction
 Energy of activation, is the energy difference
between that of the reactants and a T2
high-energy intermediate (transition state),
that occurs during the formation of the product. Activation energy of
enzyme catalyzed
reaction
 To allow a reaction to proceed rapidly
, the enzyme provides an alternate A
reaction pathway with a lower free
energy of activation than that of G
the un-catalyzed reaction. B

B- Precise arrangement of chemical groups at the active site

 The amino acids at the active site are arranged in a very precise manner so that
only specific substrate or inter-related substrates can bind at the active site.
 Also, the shape and the chemical environment inside the active site permit a
chemical reaction to proceed more easily.
 Usually serine, histidine, cysteine, aspartate and glutamate residues make up
active site.

Factors Affecting the Rate of Enzyme Catalyzed Reaction

1- Substrate Concentration [S]

2- Enzyme Concentration (E)
3- Concentration of inhibitors
4- Concentration of Cofactors [C]
5- Temperature
6- PH

In studying the effect of any of the factors on enzyme catalysis, only one factor has to
be varied at a time, all other factors being kept constant. The rate of the reaction should
always be measured at the very beginning of the reaction, the so-called initial velocity
(V0 or Vi ).
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1- Effect of Substrate Concentration [S]
 If all other conditions are kept constant, the velocity of the reaction increases as
the substrate concentration [S] increases up to point where the enzyme is said to
be saturated, the measured initial velocity or Vi increases to a maximum value
Vmax.
 The substrate concentration that produces half the maximal velocity is termed
Michaelis constant or Km.
 Smaller Km reflects higher affinity of the enzyme for its substrate and vice
versa.

Michaelis-Menten Equation
The Michaelis-Menten equation describes the behavior of many enzymes as substrate
concentration is changed.
Vmax [S]
Vi =
Km + [S]

Lineweaver-Burk Plot or Double-Reciprocal Plot

The curve that describes the effect of substrate concentration is hyperbolic and it is
quite difficult to estimate the Vmax as the value is never reached with any finite
substrate concentration. This in turn makes it difficult to determine the Km value. It is
easier to work with a straight line than a curve. One can transform the equation of
hyperbola into an equation of a straight line by taking the reciprocal of both sides to
yield the following:
1 Km 1 1
= Vmax
+
Vi ]S[ Vmax

Lineweaver-Burk or double-reciprocal plot

Vi /1
Vmax Slope = Km / Vmax

Vmax 1/2
Intercept on x-axis Intercept on y-axis
Km /1- = Vmax /1 =

Km1 Km2 ]S[ 0 ]S[ /1

5- Effect of Temperature
 At 0 °C enzyme action virtually stops due to the inhibition of movement and
collision between the substrate and enzyme molecules.
 As the temperature rises the velocity of the reaction increases due to increased
kinetic energy of the molecules and increased collision between substrate and
enzyme molecules, increasing the formation of ES complex.
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 The increase in the velocity of the reaction continues up to a point, “the
optimum temperature”, beyond which any further increase in temperature
causes a decrease in the reaction rate.
 For most enzymes, the activity virtually stops at about 70 °C, due to denaturation
of the enzyme protein, disrupting the organization of the catalytic site.
 The optimum temperature for most animal enzymes is about 37°C, while that of
most plant enzymes is about 50 °C.

6- Effect of pH
 Each enzyme has an optimum pH at which it shows maximal activity.
Activity decreases as we go away from the optimum pH, it virtually stops
about 2 units of pH above or below this pH.
 Slight changes in pH causes marked changes in enzyme activity due to
alteration of the charges on the substrate and on the catalytic site of the
enzyme.
 Extreme changes of pH cause denaturation and irreversible inhibition of
enzyme action.
 Most enzymes have an optimum pH between 5 and 9. There are some
exceptions, 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

Inhibition of Enzyme Activity

Any substance that can diminish the velocity of an enzyme-catalyzed reaction is called
an inhibitor. Enzyme inhibition can be either reversible or irreversible.

I- Reversible Enzyme Inhibition

Most common types:
A. Competitive inhibition
B. Allosteric inhibition.
A- Competitive Inhibitors (Substrate analogue inhibitors)
 A competitive inhibitor is structurally similar to that of substrate. Hence, it
competes with substrate to bind reversibly at active or catalytic site.
 No Effect on Vmax: The effect of a competitive inhibitor is reversed by
increasing [S]. At a sufficiently high substrate concentration, the reaction
velocity reaches the Vmax observed in the absence of inhibitor.
 Increase of Km: A competitive inhibitor increases the apparent Km for a given
substrate. This means that, in the presence of a competitive inhibitor, more
substrate is needed to achieve ½Vmax.
Competitive inhibitor
Vi /1
Competitive inhibitor No inhibitor No inhibitor
Vmax

Vmax 1/2
Intercept on x-axis Intercept on y-axis
Km /1- = Vmax /1 =

Km1 Km2 ]S[ 0 ]S[ /1

Lineweaver-Burk or double-reciprocal plot
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Examples for competitive inhibitors

1-Allopurinol is a drug used in the treatment of gout.

i. Gout is due to excessive production of uric acid.
ii. Xanthine oxidase is an enzyme involved in the formation of uric acid from
hypoxanthine.
iii. Allopurinol is a structural analog of hypoxanthine.
iv. Allopurinol blocks the formation of uric acid by inhibiting the enzyme xanthine
oxidase.

Hypoxanthin Allopurinol
e

2-Sulfonamides are used in the treatment of bacterial infections.

i. Bacteria synthesize folic acid from p-aminobenzoic acid (PABA).
ii. Since these sulfonamide drugs contain sulfanilamide a structural analog of
PABA, when used as chemotherapeutic agent, it blocks the synthesis of folic
acid in bacteria which is essential for bacterial multiplication (sulfonamides act
as a bacteriostatic agent).
iii. Sulfonamides act as a competitive inhibitor for the enzyme involved in the
formation of folic acid using PABA as substrate.
Precursor Folic acid Bacterial growth
Sulfonamide Block

H 2N COOH H 2N SO2N
PABA Sulfanilamide
3- Dicumarol and warfarin are used as anticoagulants because they are structurally
similar to vitamin K (required for activation of blood clotting factors).

4- Statins are competitive inhibitors of the key enzyme of cholesterol synthesis

(HMG-CoA reductase) thereby lowering plasma cholesterol levels.

B- Allosteric Inhibitors
 They are usually small organic molecules that bind to a specific site away from
the catalytic site and produce conformational changes in protein structure that
lead to decrease activity of the enzyme e.g. ATP for phosphofructokinase-1.
 Allosteric inhibitors decrease the affinity of the enzyme to its substrate (increase
Km value), decrease the maximal catalytic activity (decrease Vmax) or both.

Feedback Inhibition: It is the inhibition of the activity of an enzyme in a pathway by

the end products of this pathway.
It may occur through binding of the end product with an allosteric site present on the
regulatory key enzyme. This usually occurs at the earliest irreversible step unique to
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that sparticular pathway. This prevents accumulation of unwanted amounts of the
metabolic end products.

II- Irreversible Enzyme Inhibition:

A- Inhibitors that exert their effects on enzymes
1.Inhibitors that denature proteins (These include strong acids, alkalis,
alcohols and salts of heavy metals).
2.Antienzymes: Antienzyme is specific for the enzyme that binds with it and
produces its inactivation. Example: Antithrombin (activated by heparin)
inhibits blood clotting.
3.Inhibitors that block chemical groups (enzyme poisons)
a) Inhibitors of the sulfhydryl group (SH group)
i. Oxidizing agents. Example: Hydrogen peroxide (H2O2)
ii. Salts of heavy metals. Example: Hg2+ combine with the negatively
charged sulfur of the SH group (Enz-S- Hg -S-Enz).

b) Inhibitors that block hydroxyl groups (OH group)

Aspirin produces acetylation of the hydroxyl group of serine at the active
site of cyclooxygenase enzyme (responsible for prostaglandin synthesis),
explaining the anti-inflammatory and antipyretic actions of aspirin.

B- Inhibitors that exert their effects on cofactors or prosthetic groups

1. Fluoride blocks the action of enzymes, which require Ca2+ & Mg2+ ions by
chelating these ions in the form of salts e.g. chelation of Mg2+ in case of
enolase (an enzyme of glucose oxidation), results in inhibition of the
enzyme.
2. Cyanide and carbon monoxide inhibit the activity of cytochrome oxidase
an enzyme of respiratory chain by blocking the iron of heme.

Regulation of Enzyme Activity

It is important to control the rate of different metabolic reactions to maintain cellular

structures and functions under different conditions. Regulation of enzyme activity is
achieved through different mechanisms.
I. Changing the E absolute amount & II. Changing the catalytic activity of the E.
I- Changing the Absolute Amount of the Enzyme Present
The amount of the enzyme present is determined by its rate of synthesis and rate of
degradation.

1- Control of Enzyme Synthesis

This is mainly performed through inducers and repressors.
Inducers are substances, which stimulate gene expression into proteins. In case of
enzymes, the inducers may be the substrate of the enzyme or hormones.
Repressors are substances, which inhibit gene expression into proteins. In case of
enzymes, the repressor may be a metabolic product of the enzyme or hormones.

2- Control of Enzyme Degradation

 Enzyme
Thiss is performed by controlling the rate of synthesis or activity of the enzyme
responsible for their degradation

II. Changing the Catalytic Activity of the Enzyme.

1- Activation of Zymogens (Proenzymes)
 Many enzymes are formed in the form of proenzymes or zymogens. In this
form they are inactive. Activation requires proteolysis (removal of a part of the
polypeptide chain which masks the active site or substrate site).
 Many of these enzymes after activation can activate its zymogen in a process
termed autocatalytic activation (autocatalysis).

2- Allosteric Modifiers (Inhibitors and Activators)

 Allosteric inhibition is explained before.
The binding of an allosteric activator with the allosteric site produces
conformational changes in the protein structure of the enzyme, which result in
increased velocity of the reaction.
Many cellular metabolic reactions are controlled in this way e.g. AMP acts as an
allosteric activator for the phosphofructokinase-1 (PFK-1).
Allosteric activator increases the affinity of the enzyme to its substrate (decrease
Km value), increases the maximal catalytic activity (increase Vmax) or both.

3- Covalent Modification
 Phosphorylation and dephosphorylation: Many enzymes are activated by
phosphorylation and inactivated by dephosphorylation and vice versa.
 This means that the enzyme is present in two interconvertible forms
(phosphorylated and dephosphorylated). The phosphate groups are usually
attached to the hydroxyl group of amino acid residues (mainly serine or
tyrosine) present in the polypeptide chain of the enzyme.

Isoenzymes (Isozymes)
These are a group of enzymes which are characterized by the following:
1. They catalyze the same reaction.
2. They have different polypeptide chains which are produced by different genes.
3. They are separated by electrophoresis (have different migration rate).
4. They have different affinity to the substrate.
5. They are usually affected in different ways by the different activators and
inhibitors.
6. They are present in the same (different compartments) or different cells.
Examples for isozymes include the following:
I- Lactate dehydrogenase (LDH)
It is tetrameric enzyme. It is formed of four protomers (subunits) of two types H and
M. The tetrameric molecule is the only active form of the enzyme. The different
subunits are combined to form five isozymes as follows:
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Isozymes of LDH Type of subunits

1- I1 (H4 or HHHH)
2- I2 (H3M1 or HHHM)
3- I3 (H2M2 or HHMM)
4- I4 (H1M3 or HMMM)
5- I5 (M4 or MMMM)

H and M subunits are synthesized by distinct genes and are differentially expressed in
different tissues. Estimation of LDH isozymes in plasma is of clinical importance.
Isozyme 1 is mainly of cardiac origin and its level in plasma increases in cases of
myocardial infarction.
Isozyme 5 increases in plasma in cases of liver diseases (hepatic origin) or muscle
diseases (muscular origin).

II- Creatine Kinase (CK) or Creatine Phosphokinase (CPK)

It is a dimer formed of two subunits termed M or B. It has three isozymes as follows:
1- CK1 (or CK-BB) present in brain tissues and its plasma level increases in case
of brain infarction.
2- CK2 (or CK-MB) present in cardiac muscles more than in skeletal muscles, so
its plasma level increases markedly in myocardial infarction.
3- CK3 (or CK-MM) present in skeletal muscles more than in cardiac muscles, so
its plasma level increases markedly in muscle diseases.

Clinically Important Enzymes and Diagnostic Applications

Principle Sources of
Clinical application Principal Source Enzyme
cancer prostate Red cells and prostate Acid phosphatase
Alanine
Hepatic parenchymal diseases Liver
aminotransferase
Alkaline
Liver, bone, intestinal
Bone diseases, hepatobiliary diseases phosphatase
mucosa and placenta
Parotitis Salivary glands and
Amylase
Pancreatitis pancreas
Hepatic parenchymal disease, muscle Liver, skeletal muscle and Aspartate
and cardiac diseases heart aminotransferase
Muscle diseases and myocardial Creatine kinase
Skeletal muscle, and heart
infarction
Hemolysis, Heart, liver, skeletal
Lactate
hepatic parenchymal diseases, tumor muscle, erythrocytes,
dehydrogenase
marker platelets, lymph nodes
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