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: Reversible inhibition, Irreversible inhibition
and Allosteric inhibition

1. Reversible inhibition: The inhibitor binds non-covalently with enzyme and the enzyme inhibition can
be reversed if the inhibitor is removed. The reversible inhibition is further sub-divided into
– Competitive inhibition
– Non-competitive inhibition
– Un-Competitive inhibition
l. Competitive inhibition : The inhibitor (l) which closely resembles the real substrate (S) is regarded
as a substrate analogue. The inhibitor competes with substrate and binds at the active site of
the enzyme but does not undergo any catalysis. As long as the competitive inhibitor holds the
active site, the enzyme is not available for the substrate to bind.

• The relative concentration of the substrate and inhibitor and their respective affinity with the
enzyme determines the degree of competitive inhibition. The inhibition could be overcome by
a high substrate concentration. ln competitive inhibition, the Km value increases whereas
Vmax remains unchanged.
• The enzyme succinate dehydrogenase (SDH) is a classical example of competitive inhibition with
succinic acid as its substrate. The compounds, namely, 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.

• Methanol is toxic to the body when it is converted to formaldehyde by the enzyme alcohol
dehydrogenase (ADH). Ethanol can compete with methanol for ADH. Thus, ethanol can be used in
the treatment of methanol poisoning.

• Antimetabolites : These are the chemical compounds that block the metabolic reactions by their
inhibitory action on enzymes. Antimetabolites are usually structural analogues of substrates and
thus are competitive inhibitors. They are in use for cancer therapy, gout etc. The term antivitamins
is used for the antimetabolites which block the biochemical actions of vitamins causing deficiencies,
e.g. sulphonilamide, dicumarol.

• p-Amino benzoic acid is one of the important components of folic acid (vitamin). The derivative
(coenzymic) form of this vitamin is tetrahydrofolic acid. The sulfanilamide and its derivatives act as
competitive inhibitor of the enzyme involved in the incorporation of p-amino benzoic acid into folic
acid. The enzyme is camouflaged and sulfanilamide gets incorporated and non-functional molecule
is obtained. This slowly leads to the death of microorganism. Thus such drugs are bacteriostatic.
ll. Non-competitive inhibition: The inhibitor binds at a site other than the active site on the enzyme
surface. This binding impairs the enzyme function. For non-competitive inhibition, the Km value
is unchanged while Vmax is lowered.

• The inhibitor has no structural resemblance with the substrate. However, there usually exists a
strong affinity for the inhibitor to bind at the second site. In fact, the inhibitor does not interfere
with the enzyme-substrate binding. But the catalysis is prevented, possibly due to a distortion in
the enzyme conformation.

• The inhibitor generally binds with the enzyme as well as the ES complex.

• Heavy metal ions (Ag+, Pb2+, Hg2+ etc.) can non-competitively inhibit the enzymes by binding with
cysteinyl sulfhydryl groups. Heavy metals also lead to the formation of covalent bonds with
carboxyl groups and histidine, often resulting in irreversible inhibition.
III. Uncompetitive Inhibition: Compounds that reversibly combine with only the ES complex but not
the free enzyme are called uncompetitive inhibitors. The inhibitor is not overcome by high
substrate concentration. And substrate binds more tightly/effectively to the enzymes in the
presence of inhibitor.

• The Vmax and Km of the enzyme changes with uncompetitive inhibitor, both will decrease.
 2. lrreversible inhibition
• The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible. These
inhibitors are usually toxic poisonous substances.

• 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.

• Diisopropyl fluorophosphate (DFP) is a nerve gas developed by the Germans during Second
World War. DFP irreversibly binds with enzymes containing serine at the active site, e.g. serine
proteases, acetylcholine esterase. The penicillin antibiotics act as irreversible inhibitors of serine
- containing enzymes, and block the bacterial cell wall svnthesis.

• Many organophosphorus insecticides like melathion are toxic to animals (including man) as they
block the activity of acetylcholine esterase (essential for nerve conduction), resulting in paralysis
of vital body functions.

• lrreversible inhibitors are frequently used to identify amino acid residues at the active site of the
enzymes, and also to understand the mechanism of enzyme action.
Suicide inhibition
• Suicide inhibition is a specialized form of irreversible inhibition. ln this case, the original
inhibitor (the structural analogue/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. This is in contrast to the original inhibitor which binds reversibly.

• A good example of suicide inhibition is allopurinol (used in the treatment of gout).

Allopurinol, an inhibitor of xanthine oxidase, gets converted to alloxanthine, a more effective
inhibitor of this enzyme.

• The use of certain purine and pyrimidine analogues in cancer therapy is also explained on the
basis of the suicide inhibition. For instance, S-fluorouracil gets converted to
fluorodeoxyuridylate which inhibits the enzyme thymidylate synthase, and thus nucleotide
synthesis.
 Enzyme specificity
• Enzymes are highly specific in their action when compared with the chemical catalysts. The
occurrence of thousands of enzymes in the biological system might be due to the specific nature
of enzymes. Specificity is a characteristic property of the active site. Three types of enzyme
specificity are well-recognised:
1. Stereospecificity
2. Reaction specificity
3. Substrate specificity

1. Stereospecificity or optical specificity: Stereoisomers are the compounds which have the same
molecular formula, but differ in their structural configuration. The enzymes act only on one
isomer and, therefore, exhibit stereospecificity. e.g. L-amino acid oxidase and D-amino acid
oxidase act on L- and D-amino acids respectively; Hexokinase acts on D-hexoses; Glucokinase on
D-glucose; Amylase acts on α-glycosidic linkages; Cellulase cleaves β-glycosidic bonds.
•The class of enzymes belonging to isomerases do not exhibit stereospecificity, since they are
specialized in the interconversion of isomers.

2. Reaction specificity: The same substrate can undergo different types of reactions, each catalysed by
a separate enzyme and this is referred to as reaction specificity. An amino acid can undergo
transamination, oxidative deamination, decarboxylation, racemization etc. The enzymes however,
are different for each of these reactions.

3. Substrate specificity: The substrate specificity varies from enzyme to enzyme. lt may be either
absolute, relative or broad.
• Absolute substrate specificity: Certain enzymes act only on one substrate e.g. glucokinase acts
on glucose to give glucose-6-phosphate, urease cleaves urea to ammonia and carbon dioxide.

• Enzyme is specific only for succinate and leads to the formation of fumerate i.e. only the trans
form but not the maleic acid (cis form).

• Absolute group specificity: Many enzymes can act on different substrates having same groups
within them (point of attack on them). Example alcohol dehydrogenase – oxidizes both
methanol and ethanol, which have common hydroxyl group.
• Relative substrate specificity: Some enzymes act on structurally related substances. This, in
turn, may be dependent on the specific group or a bond present. E.g: The action of trypsin is a
good example for group specificity. Trypsin hydrolyses peptide linkage involving arginine or
lysine. Chymotrypsin cleaves peptide bonds attached to aromatic amino acids (phenylalanine,
tyrosine and tryptophan). Glycosidases acting on glycosidic bonds of carbohydrates, lipases
cleaving ester bonds of lipids etc.
• Broad specificity: Some enzymes act on closely related substrates which is commonly known
as broad substrate specificity, e.g. hexokinase acts on glucose, fructose/mannose and
glucosamine. It is possible that some structural similarity among these compounds makes
them a common substrate for the enzyme hexokinase.
 Cofactors
• A large number of enzymes requires additional non-protein components for carrying out their
catalytic activity, which are called cofactors. They have been divided into three classes.
– Prosthetic group
– Coenzyme
– Metal ions

• Cofactors are generally stable to heat, whereas protein part of the enzyme loses activity on
heating.

• The catalytically active enzyme-cofactor complex is called the holoenzyme. When the cofactor is
removed, the remaining protein, which is catalytically inactive by itself is called an apo-enzyme.

• When the cofactor is very tightly bound to the enzyme molecule, it is called a prosthetic group.
Example: Acetyl CoA carboxylase contains biotin as a prosthetic group as it is covalently bound
to ε-amino group of lysine by a covalent bond.

• Cofactors in some enzymes, when loosely held by weak forces and can be easily removed by
dialysis, are called as coenzymes. Example Pyruvate dehydrogenase contains NAD + as coenzyme.

• Some enzymes contain metal ions. They may either participate in catalytic action or may hold
the protein in proper conformation. Example, Alcohol dehydrogenase contains Zn ++, Tyrosinase
contains Cu++ and ATPase contains Na+.
 Coenzymes
• The non-protein, organic, Iow molecular weight and dialysable substance associated with
enzyme function is known as coenzyme.

• Coenzymes are second substrates : Coenzymes are often regarded as the second substrates or
co-substrates, since they have affinity for the enzyme comparable with that of the substrates.
Coenzymes undergo alterations during the enzymatic reactions, which are later regenerated.
This is in contrast to the substrate which is converted to the product.

• Coenzymes participate in various reactions involving transfer of atoms or groups like hydrogen,
aldehyde, keto, amino, acyl, methyl, carbon dioxide etc. Coenzymes play a decisive role in
enzyme function.

• Coenzymes from B-complex vitamins: Most of the coenzymes are the derivatives of water
soluble B-complex vitamins. In fact, the biochemical functions of B-complex vitamins are
exerted through their respective coenzymes.

• Non-vitamin coenzymes : Not all coenzymes are vitamin derivatives. There are some other
organic substances, which have no relation with vitamins but function as coenzymes. They may
be considered as non-vitamin coenzymes e.g. ATP, CDP, UDP etc.
• Nucleotide coenzymes: Some of the coenzymes possess nitrogenous base, sugar and phosphate.
Such coenzymes are, therefore, regarded as nucleotides e.g. NAD +, NADP+, FMN, FAD, coenzyme
A etc.

• Coenzymes do not decide enzyme specificity: A particular coenzyme may participate in catalytic
reactions along with different enzymes. For instance, NAD + acts as a coenzyme for lactate
dehydrogenase and alcohol dehydrogenase. In both the enzymatic reactions, NAD+ is involved in
hydrogen transfer. The specificity of the enzyme is mostly dependent on the apoenzyme and not
on the coenzyme.
 Mechanism of enzyme action
• For any chemical reaction, three criteria must occur:
– The reactants, called the substrates must collide.
– The molecular collision must occur in correct orientation.
– The reactants must have sufficient energy (activation energy)

• For enzyme catalyzed reaction, both higher probability of correct orientation of reactants and
lower activation energy are observed.

• The conversion of reactants to products in any chemical reaction is accompanied by a continuous

change of energy. Each combination of reactant has a defined energy.
• In a ground state, that is, at the lowest energy and most stable confirmation, the bond length,
bond angles and electron distribution of the reactants are at their equilibrium values.

• The energy of the system increases as the reactants approach each other and begin to undergo a
chemical reaction. At the point called transition state of the reaction, the energy reaches the
maximum. In this transition state, the bond length, bond angles, electron distribution of the
reactants are distorted to some higher energy configuration.

• As the reaction continues, the energy of the system decreases, until it reaches a new minimum in
the product.

• Activation energy is the energy difference between the ground state of the reactant and the
transition state. The magnitude of activation energy influences the rate of reaction, the higher
the activation energy, or barrier, the slower the reaction. By lowering down the activation energy
for the reaction it catalyzes, an increase in rate of reaction is observed.

• Enzymes lower the activation energy by forming ES complex i.e. more stable than the isolated
enzyme and its substrate. The increased stabilization is due to favored interaction between
them.

• The energy of transition is lowered because, the enzyme interacts more strongly with substrate
when the substrate is in a conformation that is closer to the transition state than to the ground
state. Enzyme binding stabilizes the transition state, thereby, decreasing the activation energy of
the reaction and decreasing the reaction rate.
 Mechanism of enzyme-substrate complex formation
Lock and key model or Fischer's template theory

• This theory was proposed by a German

biochemist, Emil Fischer. This is in fact the
very first model proposed to explain an
enzyme catalysed reaction.

• According to this model, the structure or

conformation of the enzyme is rigid.

• The substrate fits to the binding site (now

active site) just as a key fits into the proper
lock or a hand into the proper glove.

• Thus the active site of an enzyme is a rigid

and pre-shaped template where only a
specific substrate can bind.

• This model does not give any scope for the

flexible nature of enzymes, hence the model
totally fails to explain many facts of
enzymatic reactions, the most important
being the effect of allosteric modulators.
Induced fit theory or Koshland's model
• Koshland, in 1958, proposed a more acceptable and realistic model for enzyme substrate complex
formation.

• As per this model, the active site is not rigid and pre-shaped. The essential features of the substrate
binding site are present at the nascent active site. The interaction of the substrate with the enzyme
induces a fit or a conformation change in the enzyme, resulting in the formation of a strong substrate
binding site.

• Further, due to induced fit, the appropriate amino acids of the enzyme are repositioned to form the
active site and bring about the catalysis.

• Induced fit model has sufficient experimental evidence from the X-ray diffraction studies.

• Koshland's model also explains the action of allosteric modulators and competitive inhibition on
enzymes.

Substrate strain theory

• In this model, the substrate is strained due to the induced conformation change in the enzyme.

• It is also possible that when a substrate binds to the preformed active site, the enzyme induces a
strain to the substrate. The strained substrate leads to the formation of product.

• ln fact, a combination of the induced fit model with the substrate strain is considered to be operative
in the enzymatic action.
 MECHANISM OF ENZYME CATATYSIS
• The formation of an enzyme-substrate complex (ES) is very crucial for the catalysis to occur, and
for the product formation.

• lt is estimated that an enzyme catalysed reaction proceeds 10 6 to 1012 times faster than a
noncatalysed reaction. The enhancement in the rate of the reaction is mainly due to four
processes:
– Acid-base catalysis
– Substrate strain
– Covalent catalysis
– Entropy effects

1 . Acid-base catalysis : Role of acids and bases is quite important in enzymology.

• At the physiological pH, histidine is the most important amino acid, the protonated form of which
functions as an acid and its corresponding conjugate as a base.

• The other acids are –OH group of tyrosine, -SH group of cysteine, and ε-amino group of lysine. The
conjugates of these acids and carboxyl ions (COO -) function as bases.

• Ribonuclease which cleaves phosphodiester bonds in a pyrimidine loci in RNA is a classical

example of the role of acid and base in the catalysis.
2. Substrate strain:
• lnduction of a strain on the substrate occurs during ES formation. During the course of strain
induction, the energy level of the substrate is raised, leading to a transition state.

• The mechanism of lysozyme (an enzyme of tears, that cleaves β-1,4 glycosidic bonds) action is
believed to be due to a combination of substrate strain and acid-base catalysis.

3. Covalent catalysis:
• In the covalent catalysis, the negatively charged (nucleophilic) or positively charged (electrophilic)
group is present at the active site of the enzyme. This group attacks the substrate that results in
the covalent binding of the substrate to the enzyme.

• In the serine proteases (so named due to the presence of serine at active site), covalent catalysis
along with acid-base catalysis occur, e.g. chymotrypsin, trypsin, thrombin etc.

4. Entropy effect :
• Entropy is a term used in thermodynamics. It is defined as the extent of disorder in a system. The
enzymes bring about a decrease in the entropy of the reactants. This enables the reactants to
come closer to the enzyme and thus increase the rate of reaction.

In the actual catalysis of the enzymes, more than one of the processes, acid-base catalysis, substrate
strain, covalent catalysis and entropy are simultaneously operative. This will help the substrate(s)
to attain a transition state leading to the formation of products.
 THERMODYNAMICS OF ENZYMATIC REACTIONS
The enzyme catalysed reactions may be broadly grouped into three types based on thermodynamic
(energy) considerations.

1. lsothermic reactions: The energy exchange between reactants and products is negligible. e .g.
glycogen phosphorylase

2. Exothermic (exergonic) reactions : Energy is liberated in these reactions e.g. urease

3. Endothermic (endergonic) reactions : Energy is consumed in these reactions e.g. glucokinase

 Units of enzyme activity
• Enzymes are never expressed in terms of their concentration (as mg or pg etc.), but are expressed
only as activities.

• Various methods have been introduced for the estimation of enzyme activities. In fact, the
activities have been expressed in many ways, like King-Armstrong units, Somogyi units, Reitman-
Frankel units, spectrophotometric units etc.

Katal
• In order to maintain uniformity in the expression of enzyme activities (as units) worldover, the
Enzyme Commission of IUB has suggested a unit- namely katal (abbreviated as kat).

• One kat denotes the conversion of one mole of substrate per second (mol/sec). Activity may also
be expressed as millikatals (mkat), microkatals (μkat) and so on.

International Units (IU)

• Some workers prefer to use standard units or Sl units (System International). One Sl unit or
International Unit (lU) is defined as the amount of enzyme activity that catalyses the conversion
of one micromol of substrate per minute.

• Sl units and katal are interconvertible.

 Regulation of enzyme activity in the living
system
In biological system, regulation of enzyme activities occurs at different stages in one or more of the
following ways to achieve cellular economy:

1. Allosteric regulation

2. Activation of latent enzymes

3. Compartmentation of metabolic pathways

4. Control of enzyme synthesis

5. Enzyme degradation

6. lsoenzymes
 Allosteric regulation and 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.

• Allosteric effectors: 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.

• Classes of allosteric enzymes: Allosteric enzymes are divided into two classes based on the
influence of allosteric effector on K m and Vmax.

– K-class of allosteric enzymes, the effector changes the Km and not the Vmax. Double reciprocal
plots, similar to competitive inhibition are obtained e.g. phosphofructokinase.

– V-class of allosteric enzymes, the effector alters the V max and not the Km. Double reciprocal
plots resemble that of non-competitive inhibition e.g. acetyl CoA carboxylase.
 Conformational changes in allosteric
enzymes
• Most of the allosteric enzymes are oligomeric in nature. The subunits may be identical or
different. The non-covalent 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 (Fig.6.13).

• In the concerted model, 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 favour T state whereas activators and substrates favour R state. The substrate
can bind only with the R form of the enzyme. The concentration of enzyme molecule in the R
state increases as more substrate is added, therefore the binding of the substrate to the allosteric
enzyme is said to be cooperative.

• Allosteric enzymes give a sigmoidal curve (instead of hyperbola) when the velocity (v) versus
substrate(S) concentration are plotted (Fig.6.14)
 Feedback regulation
• The process of inhibiting the first step by the final product, in a series of enzyme catalysed
reactions of a metabolic pathway is referred to as feedback regulation.

• A is the initial substrate B, C, and D are the intermediates and E is the end product, in a pathway
catalysed by four different enzymes (e1, e2, e3, e4). The very first step (A →B by the enzyme e1 )
is the most effective for regulating the pathway, by the final end product E. This type of control is
often called negative feedback regulation since increased levels of end product will result in its
(e1) decreased synthesis. This is a real cellular economy to save the cell from the wasteful
expenditure of synthesizing a compound which is already available within the cell.

• Feedback inhibition or end product inhibition is a specialised type of allosteric inhibition

necessary to control metabolic pathways for efficient cellular function.

• Aspartate transcarbamoylase (ATCase) is a good example of an allosteric enzyme inhibited by a

feedback mechanism. ATCase catalyses the very first reaction in pyrimidine biosynthesis.

• Carbamoyl phosphate undergoes a sequence of reactions for synthesis of the end product, CTP.
When CTP accumulates, it allosterically inhibits the enzyme aspartate transcarbamoylase by a
feedback mechanism.
 Activation of latent enzymes
• Latent enzymes, as such, are inactive. Some enzymes are synthesized as Proenzymes or
zymogens which undergo irreversible covalent activation by the breakdown of one or more
peptide bonds.

• For instance, proenzymes-namely chymotrypsinogen, pepsinogen and plasminogen, are

respectively- converted to the active enzymes chymotrypsin, pepsin and plasmin. Certain
enzymes exist in the active and inactive forms which are interconvertible, depending on the
needs of the body.

• The interconversion is brought about by the reversible covalent modifications, namely

phosphorylation and dephosphorylation, and oxidation and reduction of disulfide bonds.

• Glycogen phosphorylase is a muscle enzyme that breaks down glycogen to provide energy.
This enzyme is a homodimer (two identical subunits) and exists in two interconvertible forms.

• Phosphorylase b (dephosphoenzyme) is inactive which is converted by phosphorylation of

serine residues to active form phosphorylase a. The inactive enzyme phosphorylase b is
produced on dephosphorylation as illustrated below.
• There are some enzymes which are active in dephosphorylated state and become inactive when
phosphorylated e.g. glycogen synthase, acetyl CoA carboxylase.

• A few enzymes are active only with sulfhydryl (-SH) groups, e.g: succinate dehydrogenase, urease.
Substances like glutathione bring about the stability of these enzymes.
 Compartmentation
• There are certain substances in the body (e.g., fatty acids, glycogen) which are synthesized and
also degraded. There is no point for simultaneous occurrence of both the pathways.

• Generally, the synthetic (anabolic) and breakdown (catabolic) pathways are operative in
different cellular organelles to achieve maximum economy. For instance, enzymes for fatty acid
synthesis are found in the cytosol whereas enzymes for fatty acid oxidation are present in the
mitochondria.

• Depending on the needs of the body – through the mediation of hormonal and other controls -
fatty acids are either synthesized or oxidized.
 Control of enzyme synthesis
• Most of the enzymes, particularly the rate limiting ones, are present in very low concentration.
Many rate Iimiting enzymes have short half-lives. This helps in the efficient regulation of the enzyme
levels.

• There are two types of enzymes-(a) Constitutive enzymes (house-keeping enzymes)-the levels of
which are not controlled and remain fairly constant. (b) Adaptive enzymes-their concentrations
increase or decrease as per body needs and are well-regulated.

• Induction and repression: The term induction is used to represent increased synthesis of enzyme
while repression indicates its decreased synthesis. Induction or repression which ultimately
determines the enzyme concentration at the gene level through the mediation of hormones or other
substances.

• Examples of enzyme induction : The hormone insulin induces the synthesis of glycogen synthetase,
glucokinase, phosphofructokinase and pyruvate kinase. All these enzymes are involved in the
utilization of glucose. The hormone cortisol induces the synthesis of many enzymes e.g. pyruvate
carboxylase, tryptophan oxygenase and tyrosine aminotransferase.

• Examples of repression : In many instances, substrate can repress the synthesis of enzyme. Pyruvate
carboxylase is a key enzyme in the synthesis of glucose from non-carbohydrate sources like pyruvate
and amino acids. lf there is sufficient glucose available, there is no necessity for its synthesis. This is
achieved through repression of pyruvate carboxylase by glucose.
 Enzyme degradation
• Enzymes are not immortal.

• There is a lot of variability in the half-lives of individual enzymes. For some, it is in days while
for others, it is in hours or in minutes, e.g. LDH 4- 5 to 6 days; LDH1 - 8 to 12 hours; amylase -3
to 5 hours.

• ln general, the key and regulatory enzymes are most rapidly degraded. lf not needed, they
immediately disappear and, as and when required, they are quickly synthesized.

• Though not always true, an enzyme with long half-life is usually sluggish in its catalytic
activity.
 lsoenzymes
• Multiple forms of the same enzyme will also help in the regulation of enzyme activity.

• The multiple forms of an enzyme catalyzing the same reaction are isoenzymes or isozymes.

• They, however, differ in their physical and chemical properties which include the structure,
electrophoretic and immunological properties, K m and Vmax values, pH optimum, relative
susceptibility to inhibitors and degree of denaturation.

• Many of the isoenzymes are tissue-specific.

• Many possible reasons are offered to explain the presence of isoenzymes in the living systems.
– lsoenzymes synthesized from different genes e.g. malate dehydrogenase of cytosol is
different from that found in mitochondria.

– An enzyme may be active as monomer or oligomer e.g. glutamate dehydrogenase.

– Oligomeric enzymes consisting of more than one type of subunits e.g. lactate dehydrogenase
and creatine phosphokinase.

– In glycoprotein enzymes, differences in carbohydrate content may be responsible for

isoenzymes e.g. alkaline phosphatase.
 lsoenzymes of lactate dehydrogenase (LDH)
• Among the isoenzymes, LDH has been the most thoroughly investigated. LDH whose systematic
name is L-lactate-NAD+ oxidoreductase (E.C. 1 .1.1.27) catalyses the interconversion of lactate
and pyruvate as shown below

• LDH has five distinct isoenzymes; LDH1, LDH2, LDH3, LDH4 and LDH5. They can be separated by
electrophoresis. LDHI has more positive charge and fastest in electrophoretic mobility while
LDH5 is the slowest.

• Structure of LDH isoenzymes : LDH is an oligomeric (tetrameric) enzyme made up of four

polypeptide subunits. Two types of subunits namely M (for muscle) and H (for heart) are
produced by different genes. M-subunit is basic while H subunit is acidic. The isoenzymes
contain either one or both the subunits giving LDH1 to LDH5.
 Application of enzymes
• Certain enzymes are useful as therapeutic agents, analytical reagents, in genetic manipulations
and for industrial applications (Table 6.7).

• Enzymes as therapeutic agents

1. Streptokinase prepared from streptococcus is useful for clearing the blood clots. Streptokinase
activates plasma plasminogen to plasmin which inturn, attacks fibrin to convert into soluble
products.

2. The enzyme asparaginase is used in the treatment of leukemias. Tumor 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.
Enzyrmes as analytical reagents:
• Some enzymes are useful in the clinical laboratory for the measurement of substrates, drugs,
and even the activities of other enzymes.

• The biochemical compounds (e.g. glucose, urea, uric acid, cholesterol) can be more accurately
and specifically estimated by enzymatic procedures compared to the conventional chemical
methods. A good example is the estimation of plasma glucose by glucose oxidase and
peroxidase method.

lmmobilized enzymes:
• Enzymes can be used as catalytic agents in industrial and medical applications. Some of these
enzymes are immobilized by binding them to a solid, insoluble matrix which will not affect the
enzyme stability or its catalytic activity.

• Beaded gels and cyanogen bromide activated sepharose are commonly used for
immobilization of enzymes. The bound enzymes can be preserved for long periods without
loss of activity.

• 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.
• The intensity of the blue colour depends on the concentration of glucose. Hence, the strip
method is useful for semi-quantitative estimation of glucose in urine.