ENZYMES

❑ Enzymes: Definition, properties and IUB & MB (International union of Biochemistry & Molecular biology)
classification.
❑ Factors affecting enzyme activity and mechanism of action of enzymes
❑ Enzyme inhibitors, therapeutic and pharmaceutical importance of enzymes.
Enzymes are biocatalysts – the catalysts of life. A catalyst is defined as a substance that increases the velocity or rate of a chemical
reaction without itself undergoing any change in the overall process. Enzymes may be defined as biocatalysts synthesized by living
cells. They are protein in nature (exception – RNA acting as ribozyme), colloidal and thermolabile in character, and specific in
their action.
Existence of life is unimaginable without the presence of enzymes.
E.g., Hydrolysis of proteins by a strong acid at 100°C takes at least a couple of days. The same protein is fully digested by the
enzymes in gastrointestinal tract at body temperature (37°C) within a couple of hours. This difference in the chemical reactions
taking place in the living system is due to enzymes.
Historical points:
❑ In 1836, Berzillius coined the term Catalysis.
❑ In 1878, Kunhe used the term ‘Enzyme’ to indicate the catalysis taking place in biological system.
❑ In 1883, Buchner isolated the enzyme system ‘Zymase’ from Yeast. Later, zymase was found to be a mixture of enzymes
which convert sugar to alcohol.
❑ In 1926, James Sumner isolated the enzyme urease from ‘Jack Bean’ and identified it as protein.

Properties of enzymes:
1. Enzymes are protein in nature. They give chemical reactions of proteins.
2. Enzymes exist in tertiary structure which is essential for its catalytic activity.
3. The functional unit of enzyme is called holoenzyme which is made up of two parts i.e., apoenzyme (protein part) and
coenzyme (non-protein organic part / prosthetic group).
4. They are heat labile.
5. They are water soluble.
Nomenclature and classification:
In earlier days, enzymes were named according to their discoverers and that names didn’t convey complete information about the
enzyme and its nature. In some enzymes a suffix ‘ase’ was added to the substrate for naming enzymes. These are known as trivial
names.
Enzymes were also categorised in broadly as intracellular enzymes (functional within cells where they are synthesised) and
extracellular enzymes (active outside the cell as all digestive enzymes).
International Union of Biochemistry (IUB) & Molecular Biology (MB) in 1964, gave IUB system of enzyme classification
according to the type of reactions involved in enzyme action. This system classified enzymes in to six major classes:
1. Oxidoreductases: Enzymes involved in oxidation-reduction reactions. E.g. Cytochrome oxidase, L- and D-amino acid oxidase.
2. Transferases: Enzymes that catalyse the transfer of functional group. E.g. Hexokinase, transaminase, phosphorylase etc.
3. Hydrolases: Enzymes involved in hydrolysis of various compounds. E.g., Lypase, pepsin, Urease etc.
4. Lyases: Enzymes responsible for addition or removal of water, ammonia, CO2 etc. E.g., Aldolase, fumarase etc.
5. Isomerases: Enzymes involved in isomerization reactions. E.g., Triose phosphate isomerase, phosphate hexose isomerase etc.
6. Ligases: Enzymes involved in synthetic reactions where two molecules are joined together and ATP is used. E.g., Glutamine
synthetase, succinate thiokinase etc.

Factors affecting enzyme activity:

1. Concentration of enzyme: By the increase in concentration of enzyme, velocity of enzyme
increases proportionally.
2. Concentration of substrate: By the increase in substate concentration,
velocity of reaction increases gradually but up to an extent. This factor gives a
rectangular hyperbola graph when velocity of reaction is plotted against substrate
concentration. Three phases can be observed in the graph formed as linear phase,
curve phase and unchanged phase.

3. Effect of temperature: Increase in temperature increases velocity of reaction

up to an extent when optimum temperature is reached and then declines
gradually. A bell shaped curve is obtained during this.

Note: Temperature coefficient Q10: It is defined as the increase in enzyme

velocity when temperature is increased by 10°C. For most of the enzymes, Q10 is
2 between 0°C and 40°C.

4. Effect of pH: Increase in hydrogen concentration affects enzyme velocity in a

way that a bell shaped curve is obtained. Every enzyme has an optimum pH at
which velocity is maximum. Below this pH, the activity of enzyme is lower and
above this pH at extreme, the enzyme becomes inactive.

Note: Most of the enzymes shows their optimum activity at neutral pH (6-8).
Some exceptions are also there like pepsin (1-2), acid phosphates (4-5), alkaline
phosphates (10-11).
5. Effect of product concentration: The accumulation of reaction products decreases enzyme activity as in most of the enzymes,
products form a loose complex with the active site of enzyme and hence inhibit substrate to bind at that site. This decreases the
velocity of reaction.
6. Effect of activators: Several enzymes require metallic cations like Mg++, Ca++, Zn++, K+, Na+ etc for their optimum activity.
Metals acts as activators for enzyme velocity by various mechanisms like combining with the substrate, formation of ES metal
complex, direct participation in the reaction or by bringing conformational changes in the enzyme.
Note: Several enzymes also need anions for their activation like Cl- ion is needed to activate amylase enzyme.

Enzymes activated by metal ions are categorized as follows:

1. Metal activated enzymes: The metal is not tightly held by the enzyme and can be easily exchanged with other ions. E.g.,
ATPase (Mg++ and Ca++)
2. Metalloenzymes: Enzymes hold the metal tightly which cannot be exchanged. E.g., alcohol dehydrogenase, carbonic
anhydrase, alkaline phosphatase etc. contains zinc, phenol oxidase contains copper, pyruvate oxidase contains manganese
etc.
7. Effect of time: Under ideal conditions of pH and temperature, the time required for an enzyme action is less. Variation in time
is related to the alteration in pH and temperature.
8. Effect of light and radiation: Exposure of enzymes to UV, beta, gamma and X-rays inactivates certain enzymes due to
formation of peroxidases. E.g., UV rays inhibits salivary amylase activity.

Active site of enzyme: It is the region of enzyme where substrate binds and participates in catalysis. Active site is formed because
of the tertiary conformation of enzyme.
⁂ The active site is made up of amino acids in linear sequence (primary structure). They are regarded as clefts or pockets
occupying a small region in big enzyme molecule. E.g., Enzyme lysozyme has 129 amnio acids but the active site is formed by
amino acid residues numbering 35, 52, 62, 63 and 101.
⁂ Active site is flexible in nature to promote specific binding of substrate.
⁂ Active site possess two sites, substrate binding site and catalytic site. Catalytic site is for catalysis of specific reaction.
⁂ Binding of substrate to active site is by weak non-covalent bond. Enzymes are specific in their action due to presence of active
site.
⁂ The commonly found amino acids at the active sites are serine, aspartate, histidine,
cysteine, lysine, arginine, glutamate, tyrosine etc. Among these amino acids, serine
is the most frequently found.
⁂ The substrate[S] binds the enzyme (E) at the active site to form enzyme-substrate
complex (ES). The product (P) is released after the
catalysis and the enzyme is available for reuse.

MECHANISM OF ENZYME ACTION: The main function of enzymes is to perform

catalysis and for catalysis or chemical reaction to occur on body, thew reactant must be in
activated or transition state.
*The energy required by reactants to activate/undergo the reaction is known as
activation energy. The reactants attain their activation energy on heating. The catalyst/
enzyme reduces activation energy and hence the reaction proceeds at low temperature.
The enzyme reduces activation energy of reactants in such a way that all the reaction
occurs at normal body temperature (below 40°C). Also, they only enhance the velocity of
reaction and do not alter the equilibrium constants.

Figure denotes Effect of enzyme on activation energy of a reaction (A is the substrate and
B is the product. Enzyme decreases activation energy).
*During catalysis process, the substrate(S) binds with the enzyme(E) at active site and
forms an enzyme-substrate complex (ES) which ultimately results in product formation.
*ES complex is reversible process but the formation of product is irreversible (Michaelis
Menten theory).
*Various theories have been introduced to explain the mechanism of enzyme-substrate
complex formation like lock and key model/Fischer’s template theory, induced fit
theory/Koshland’s model and substrate strain theory.

LOCK AND KEY MODEL/FISCHER’S TEMPLATE THEORY: This theory was

proposed by biochemist Emil Fischer and was the first theory proposed to explain enzyme
catalysed reactions.

“This theory states that the enzyme is rigid and substrate binds to the active site just like
key fits in to a lock.” This theory failed because it did not explained the flexible nature of
enzymes.

INDUCED FIT THEORY/KOSHLAND’S MODEL: This theory was proposed by

Koshland in 1958 and was the most acceptable model for enzyme-substrate complex
formation.
“This theory states that the binding site of enzyme is not rigid or pre-shaped, rather the
interaction of substrate to enzyme induces a conformation change in the enzyme and
results in the formation of strong substrate binding site.
*Koshland's model also explains the action of allosteric modulators and competitive
inhibition on enzymes.
SUBSTRATE STRAIN THEORY: This theory states that due to induced conformation change in the enzyme, substrate is
strained or it may be possible that when a substrate binds to preformed active site, the enzyme induces a strain to the substrate.
This strained substrate leads to the formation of product. The combination of the induced fit model with the substrate strain is
considered to be operative in the enzymatic action.

ENZYME CATALYSIS: Formation of enzyme-substrate complex is important for catalysis of enzyme and formation of product.
An enzyme catalyzed reaction proceeds 106 to 1012 times faster than a non-catalysed reaction. The enhancement in the rate of the
reaction is mainly due to four processes :
1. Acid-base catalysis
2. Substrate strain
3. Covalent catalysis
4. Entropy effects.

1. Acid-base catalysis: In this catalysis, an amino acid gets protonated (addition of a proton) to function as acid and its
corresponding conjugate as base. Mostly, at physiological pH, protonated histidine acts as acid and its conjugate as base. Other
acids like –OH group of tyrosine, -SH group of cysteine etc. also acts as acids. E.g., Ribonuclease works on acid base catalysis.
2. Substrate strain: During strain induced mechanism of ES complex formation, the energy level of substrate rises, leading to
transition state. E.g., The enzyme lysozyme works by the combination of substrate strain and acid-base catalysis.
3. Covalent catalysis: In this, a nucleophile or an electrophile is present at the active site of enzyme and attacks the substrate.
This results in the formation of covalent bond between enzyme and substrate. E.g., Enzymes like trypsin, chymotrypsin,
thrombin etc. works by covalent catalysis along with acid-base catalysis.
4. Entropy effect/covalent catalysis: In this, the substrate should come in close proximity to the enzyme for appropriate binding
and catalysis to occur. The more substrate concentration, more will be the rate of reaction, as substrate binds easily with the
enzyme and it helps in faster formation of products.
Note: During catalysis, more than one processes (Acid-base catalysis, substrate strain, covalent catalysis, and entropy catalysis)
operates simultaneously.

COENZYMES: The protein part of the enzyme is not always adequate to bring about the catalytic activity. Many enzymes
require certain nonprotein small additional factors, collectively referred to as cofactors for catalysis. The cofactors may be organic
or inorganic in nature.
• The nonprotein, organic, low molecular weight substance associated with enzyme function is known as coenzyme.
• The term prosthetic group is used when a nonprotein moiety is tightly bound to the enzyme which is not easily separable by
dialysis.
• The term activator is referred to the non-protein inorganic cofactor (like Ca2+, Mg2+, Mn2+ etc.) necessary to enhance
enzyme activity.
• Coenzymes undergo alterations during the enzymatic reactions, which are later regenerated.
• Coenzymes participate in various reactions involving transfer of atoms or groups like hydrogen, aldehyde, keto, amino, acyl,
methyl, carbon dioxide etc.
• Most of the coenzymes are the derivatives of water soluble B-complex vitamins. Coenzyme A or acetyl coenzyme A (B5),
pyridoxal 5’Phosphate (B6), Tetrahydro folic acid (B9), thyamine pyrophosphate (B1).
• Some other organic substances which function as coenzymes are considered as non-vitamin coenzymes e.g. ATP, CDP, UDP
etc.
• Some of the coenzymes possess nitrogenous base, sugar and phosphate. Such coenzymes are, therefore, regarded as nucleotides
e.g. NAD+ (B3), NADP+, FMN (B2), FAD (B2), coenzyme A, UDPG etc
• Thioredoxin is a protein that serves as a coenzyme for the enzyme ribonucleotide reductase.
• The specificity of the enzyme is mostly dependent on the apoenzyme and not on the coenzyme. E.g., NAD+ acts as a
coenzyme for lactate dehydrogenase and alcohol dehydrogenase. In both the enzymatic reactions, NAD+ is involved in
hydrogen transfer.
ENZYME INHIBITION: Enzyme inhibitor is defined as a substance which binds
with the enzyme and brings about a decrease in catalytic activity of that enzyme. The
inhibitor may be organic or inorganic in nature. There are three broad categories of
enzyme inhibition
1. Reversible inhibition.
2. Irreversible inhibition.
3. Allosteric inhibition.

1. Reversible inhibition: The inhibitor binds non-covalently with enzyme and

enzyme activity can be retained if the inhibitor is removed. It is of two types:
a. Competitive inhibition: The inhibitor closely resembles with the substrate
(substrate analogue) and binds at the active site of enzyme but does not
undergo catalysis. The relative concentration of the substrate and inhibitor and
their respective affinity with the enzyme determines the degree of competitive
inhibition. The enzyme succinate dehydrogenase (SDH) is a classical example
of competitive inhibition with succinic acid as its substrate. The compounds
like malonic acid, glutaric acid and oxalic acid, have structural similarity with
succinic acid and compete with the substrate for binding at the active site of
SDH.
b. Non-competitive inhibition: The inhibitor binds at a site other than the
active site on the enzyme surface. This binding impairs the enzyme function.
The inhibitor has no structural resemblance with the substrate. the inhibitor
does not interfere with the enzyme-substrate binding. But the catalysis is
prevented, possibly due to a distortion in the enzyme conformation.
2. Irreversible inhibition: The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible. These
inhibitors are usually toxic substances that poison enzymes. E.g., Iodoacetate is an irreversible inhibitor of the enzymes like papain
and glyceraldehyde 3-phosphate dehydrogenase. Iodoacetate combines with sulfhydryl (–SH) groups at the active site of these
enzymes and makes them inactive. The penicillin antibiotics act as irreversible inhibitors of serine – containing
enzymes, and block the bacterial cell wall synthesis.
*Suicide inhibition is a specialized form of irreversible inhibition. In this case, the original inhibitor (competitive inhibitor) is
converted to a more potent form by the same enzyme that ought to be inhibited. The so formed inhibitor binds irreversibly with the
enzyme. Example of suicide inhibition is allopurinol (used in the treatment of gout) is an inhibitor of xanthine oxidase, gets
converted to alloxanthine, a more effective inhibitor of this enzyme.

3. Allosteric inhibition: Some of the enzymes possess additional sites, known as allosteric sites (Greek : allo–other), besides the
active site. Such enzymes are known as allosteric enzymes. The allosteric sites are unique places on the enzyme molecule.
*Certain substances referred to as allosteric modulators (effectors or modifiers) bind at the allosteric site and regulate the
enzyme activity. The enzyme activity is increased when a positive (+) allosteric effector binds at the allosteric site known as
activator site. On the other hand, a negative (–) allosteric effector binds at the allosteric site called inhibitor site and inhibits
the enzyme activity.
*The noncovalent reversible binding of the effector molecule at the allosteric site brings about a conformational change in the
active site of the enzyme, leading to the inhibition or activation of the catalytic activity.
*Allosteric enzymes exist in two conformational states – the T (tense or taut) and the R (relaxed). The T and R states are in
equilibrium.
*Allosteric inhibitors favor T state whereas activators and substrates favor R state.
*The term homotropic effect is used if the substrate influences the substrate binding through allosteric mechanism, their effect
is always positive. Heterotropic effect is used when an allosteric modulator effects the binding of substrate to the enzyme.
Heterotropic interactions are either positive or negative.
Therapeutic and pharmaceutical importance of enzymes:
1. Streptokinase: Prepared from streptococcus is used for clearing blood clots. Streptokinase activates plasma plasminogen to
plasmin which, in turn, attacks fibrin to convert into soluble products.
2. Asparaginase: Used in the treatment of leukaemia. Tumour cells are dependent on asparagine of the host's plasma for their
multiplication. By administering asparaginase, the host's plasma levels of asparagine are drastically reduced. This leads to
depression in the viability of tumor cells.
3. Some enzymes are useful in the clinical laboratory for the measurement of substrates, drugs, and even the activities of other
enzymes. example is the estimation of plasma glucose by glucose oxidase and peroxidase method.
4. Some enzymes are immobilized by binding them to a solid, insoluble matrix which will not affect the enzyme stability or its
catalytic activity. The bound enzymes can be preserved for long term without loss of activity. E.g., Glucose oxidase and
peroxidase, immobilized and coated on a strip of paper, are used in the clinical laboratory for the detection of glucose in urine.

ISO-ENZYMES: The multiple forms of an enzyme catalyzing the same reaction are isoenzymes or isozymes. They differ in their
physical and chemical properties which include the structure, electrophoretic and immunological properties, Km and Vmax values,
pH optimum, relative susceptibility to inhibitors and degree of denaturation.
*Isoenzymes synthesized from different genes e.g. malate dehydrogenase of cytosol is different from that found in mitochondria.
LDH (Lactate dehydrogenase) has five distinct isoenzymes LDH1, LDH2, LDH3, LDH4 and LDH5.

Diagnostic importance of enzymes in body fluids and tissues:

➢ Urinary amylase is increased in acute pancreatitis.
➢ β-Glucuronidase is increased in the cancers of urinary bladder, pancreas etc.
➢ Lactate dehydrogenase is increased in CSF in meningitis.
➢ β-Glucuronidase activity is increased in gastric carcinoma.
➢ Glucose 6-phosphatase in liver is significantly lower in type I glycogen storage disease.