it will be helpful to various medical course students

2. INTRODUCTION
• Enzymes are the biological catalysts
which are required in almost all the
biochemical reactions in a living
system.
• Biocatalysts are designated as
enzyme since they were discovered
as the substances found in yeasts
(Greek- en=in, and zyme=yeast).
• Enzymes are biocatalysts-the
catalysts of life.
• A good teacher is always a good
catalyst in students’ life!
DIFFERENCE BETWEEN AN
ENZYME AND A CHEMICAL
CATALYST
• An enzyme catalyzed reaction has a
higher reaction rate than that of the
corresponding chemically catalyzed
reaction.
• An enzymatic reaction occurs under
relatively milder conditions, such as at
temperature near 37℃, pH near
neutrality and at atmospheric
pressure.
• Enzymes have a greater degree of
specificity for their substrates as well
as products.
• Several enzyme catalyzed reactions
are under regulatory control.

3. HISTORICAL BACKGROUND
• Berzelius in 1836 coined the term
catalysis.
• In 1878, Kuhne used the word
enzyme to indicate the catalysis
taking place in the biological
systems.
• Isolation of enzyme system from
cell-free extract of yeast was
achieved in 1883 by Buchner.
• In 1926, James Sumner first
achieved the isolation and
crystallization of the enzyme urease
from jack bean and identified it as a
protein.
CHEMICAL NATURE OF AN
ENZYME
• Most of the enzymes are protein in
nature with large molecular weight,
however, a few RNA molecules have
been shown to function as enzymes.
• Each enzyme has its own tertiary
structure and specific conformation
which is very essential for its catalytic
activity.

4. MULTIMERIC PROTEINS
• Most of the enzymes
though are simple
proteins having a single
polypeptide chain, there
are several enzymes
which have more than
one polypeptide chain.
These are called as
multimeric proteins.
• Such enzymes
(multimeric proteins) may
be found as either
oligomeric enzymes or as
multimeric complexes.
OLIGOMERIC ENZYMES
• Enzymes with two or more
subunits are called
oligomeric enzymes.
• For example, phosphorylase,
hexokinase, lactate
dehydrogenase etc.
• These occur as tetramers, i.e.
each enzyme has four
protein subunits.
• Others like
phosphofructokinase,
fructose-1-bisphosphatase
and creatine phosphokinase
are dimers, i.e. each has two
protein subunits.

5. Multienzyme complex
• Several enzymes occur in the form of a multienzyme complex, i.e. they have
more than one enzyme activities.
• Such a complex catalyzes the conversion of a substrate, in a sequential
series of reactions, to different products, e.g. pyruvate dehydrogenase
complex, α-ketoglutarate dehydrogenase complex etc. Each of these
complexes contains three enzymes.
• Fatty acid synthase is a multimeric complexes with six different enzyme
activities.

6. USEFUL TERMS
• The functional unit of the enzyme is
known as holoenzyme which is made
up of apoenzyme (the protein part) and
a coenzyme (non-protein organic part).
Holoenzyme Apoenzyme + Coenzyme
(Active enzyme) (protein part) (non-protein part)
• The term prosthetic group is used when
the non-protein moiety tightly
(covalently) binds with the apoenzyme.
• The coenzyme can be separated by
dialysis from the enzyme while the
prosthetic group cannot be.
• Some of the enzymes require the
presence of certain molecules, such as a
metal ion or an organic molecule for
their activity. The inorganic ions, such as
Mg2+, Zn2+ or Cl- required for the
catalytic activity of an enzyme are
called as cofactors.
INTRACELLULAR
LOCALIZATION
• Most of the enzymes are produced
by the cells of a particular tissue and
function within the cell. Such
enzymes are called as intracellular
enzymes, e.g. the enzymes of
glycolysis, TCA cycle and fatty acid
synthesis.
• On the other hand, there are certain
enzymes which are produced by the
cells of a particular tissue and
liberated from there for use in the
other tissues. Such enzymes are
called as extracellular enzymes, e.g.
various proteolytic enzymes like
trypsin, chymotrypsin etc. They are
secreated by the pancreatic juice for
their action in the small intestine.

7. ZYMOGEN
• Most of the intracellular enzymes
are secreated in their active form,
called ‘zymase’ form of the enzyme.
• Proteolytic enzymes on the other
hand, are usually synthesized as
somewhat larger inactive
precursors, known as ‘zymogens’.
• These molecules are stored in the
storage vesicles known as zymogen
granules, secreted as zymogens and
undergo modifications in structure,
after coming in contact with certain
activating agents.
ACUTE PANCREATITIS
• The inactivity of zymogens is crucial
because if these enzymes were
synthesized in their active forms
within the cell, this situation would
be potentially self-destructing.
• Acute pancreatitis, a painful and
sometimes fatal condition is
characterized by the premature
activation of the digestive enzymes
synthesized by this organ.
• The enzymes damage the pancreas
and spill over into the circulation.
• Pancreatic amylase is found at very
high concentration in the serum of
these patients and helps to
establish the diagnosis.

8. NOMENCLATURE OF ENZYMES
• In the early, days the enzymes were given names by
their discovers in an arbitary manner.
• Enzymes are commonly named by putting the suffix
‘-ase’ to the name of the substrate or with the
catalytic action of the enzyme.
• E.g. a lipase is an enzyme which catalyses the
hydrolysis of a lipid, a fatty acid synthase is an
enzyme that catalyzes the synthesis of a fatty acid.
• These names of the enzymes are called as trivial
names.
• While assigning a trivial name to an enzyme
however, no systematic rules are followed.

9. CLASSIFICATION OF ENZYMES
• For the rational naming of each enzyme, International Union of Biochemistry
and Molecular Biology (IUBMB) adopted a scheme and suggested functional
classification of enzymes and appointed an Enzyme Commission in 1961.
• As per IUBMB, enzymes are named with the suffix ‘ase’ with the name of the
substrate and/or the type of the reaction it catalyzes.
• Each enzyme is assigned a name and a systematic four digit enzyme code,
commonly known as Enzyme Commission number (E.C. number).
• Its first digit denotes the main class which is according to the general type of a
chemical reaction that an enzyme catalyzes.
• The second digit characterizes the sub-class, based on the nature of a
chemical group removed or transferred, or any bond split or formed.
• The third digit indicates the sub-sub-class and is used for more detailed
subdivision of the sub-class.
• The last digit of the E.C. number indicates an arbitrarily assigned serial number
to each enzyme in a sub-sub-class.
• For example, 1.1.1.1 is the enzymatic code for alcohol dehydrogenase that
catalyzes the oxidation of an alcohol to yield an aldehyde. It removes 2e- and
2H+ , and in turn reduces NAD+ .

10. • It is the first enzyme of the sub-subclass 1 (NAD+ utilizing), of the subclass 1
(dehydrogenase) and belongs to class 1 (oxidoreductase).
• Enzymes are thus classified into six major classes according to the nature of a
chemical reaction they catalyze.
 Oxidoreductases
• Enzymes involved in oxidation-reduction reactions
 Transferases
• Enzymes that catalyse the transfer of functional groups.
 Hydrolases
• Enzymes that bring about hydrolysis of various compounds.
 Lyases
• Enzymes specialized in the addition or removal of water, ammonia, CO2 etc.
 Isomerase
• Enzymes involved in all the isomerization reactions.
 Ligases
• Enzymes catalysing the synthetic reactions where two molecules are joined together
and ATP is used.

12. FACTORS AFFECTING ENZYME ACTIVITY
Fig. 5.12. Effect of enzyme concentration
1. concentration of enzyme
As the concentration of the enzyme is increased, the
velocity of the reaction proportionately increases.

13. 2. Concentration of the
substrate
• Increase in the substrate
concentration gradually
increases the velocity of enzyme
reaction within the limited range
of substrate levels.

14. 3. Effect of temperature
• Velocity of an enzyme reaction
increases with increase in
temperature up to a maximum
and then declines. A bell-shaped
curve is usually observed.
 Clinical significance
• Foods can be preserved in
refrigerators (at low
temperatures) due to reduced
bacterial enzyme activities.
• Certain surgeries are carried out
by lowering the patient’s body
temperature (induced
hypothermia), and thus the
metabolic rate.
Fig. Effect of temperature on velocity

15. Fig. Effect of pH on enzyme velocity
4. Effect of pH
The relationship between enzyme activity and pH is also represented
by a bell shaped curve which has its peak at the optimum pH. This pH
is characteristic for each enzyme. Optimum pH for most of the
intracellular enzymes is in the neutral range, i.e. around 7.0. The
optimum pH for pepsin however, is about 2.0 while for the enzymes of
the pancreatic juice, optimum pH is nearly 8.0.

16. 5. Effect of Product concentration
• The accumulation of reaction products generally decreases the enzyme
velocity.
6. Effect of activators
• Some of the enzymes require certain inorganic metallic cations like Mg2+,
Mn2+, Zn2+ etc. for their optimum activity. Rarely, anions are also needed for
enzyme activity e.g. Chloride ion (Cl-) for amylase.
• Metals function as activators of enzyme velocity through various
mechanisms- combining with the substrate, formation of ES-metal complex
etc.
7. Effect of light and radiation
• Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivates
certain enzymes due to the formation of peroxides. E.g. UV rays inhibit
salivary amylase activity.

17. ACTIVE SITE
The active site (or active centre) of an enzyme represents as the small region at
which the substrate(s) binds and participates in the catalysis.

18. Salient features of active site
1. The existence of active site is due to the tertiary structure of protein resulting
in three-dimensional native conformation.
2. The active site is made up of amino acids (known as catalytic residues) which
are far from each other in the linear sequence of amino acids (primary
structure of protein).
3. Active sites are regarded as clefts or crevices or pockets occupying a small
region in a big molecule.
4. The active site is not rigid in structure and shape. It is rather flexible to
promote the specific substrate binding.
5. Generally, the active site possesses a substrate binding site and a catalytic
site.
6. The coenzymes or cofactors on which some enzymes depend are present as a
part of the catalytic site.
7. The substrate(s) binds at the active site by weak noncovalent bonds.
8. Enzymes are specific in their function due to the existence of active sites.
9. The commonly found amino acids at the active sites are serine, aspartate,
histidine etc. Among these amino acids, serine is the most frequently found.
10. The substrate [S] binds with 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.

19. Fig. Enzymes lower the activation energy
MECHANISM OF ENZYME ACTION

20. Enzyme-substrate complex formation
• Formation of enzyme-substrate (ES) complex is the first step in enzymatic
catalysis which ultimately results in the product formation(p).
• E + S ES E + P
• Two models for substrate binding to the active site of the enzyme have been
proposed to explain the specificity that an enzyme has for its substrate.
1. Lock and Key model or Rigid Template Model of Emil Fisher
2. Induced Fit Model or Hand-in-glove Model of Daniel E Koshland
 Lock and key Model
• First model purposed to explain an enzyme catalyzed reaction.
• According to this model, the structure of 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.
• This model failed because it does not explain the changes in the enzyme
activity in the presence of allosteric modulators and also does not give any
scope for the flexible nature of the enzymes.

21. Fig. Fischer’s template theory

22. Induced Fit Model
• Daniel E Koshland in 1958
postulated that the enzymes are
flexible and shapes of the active
site can be modified by the
binding of the substrate.
• In the induced fit model, the
substrate induces a
conformational change in the
enzyme, in the same manner in
which placing a hand (substrate)
into a glove (enzyme) induces
changes in the glove’s shape.
Therefore, this model is also
known as hand-in-glove model.
• The functional groups of the
active sites are arranged in a
definite spatial configuration and
the enzyme-substrate complex is
formed by multiple bindings (such
as by covalent bonds, hydrogen
bonds and electrostatic bonds) of
the substrate with the enzyme.
Fig. Koshland’s induced fit theory

23. MECHANISM OF ENZYME CATALYSIS
• Enzymatic catalysis may take place by several processes:
1. Acid-Base catalysis
2. Covalent catalysis
3. Substrate strain
4. Proximity catalysis

24. Acid-Base catalysis
• The catalytic activity of an enzyme
may be sensitive to pH, since pH can
influence the state of protonation of
the side chain at the active site.
• At physiological pH, the protonated
form of histidine is the most
important general acid while its
conjugate base is an important
general base.
• Various biochemical reactions may
occur as a result of acid and/ or base
catalysis.
• Ribonuclease A (RNase A), which
hydrolyzes RNA to its component
nucleotides is an example of
enzymatically mediated acid-base
catalysis.
• The enzyme has two His residues, His
12 and His 119, that act in a concerted
manner as a general base and general
acid, respectively.

25. Covalent Catalysis
• In the covalent catalysis, the negatively
charged (nucleophillic) or positively
charged (electrophillic) 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.
• Functional groups in proteins, such as
the imidazole group of His, the –SH
group of cysteine, the –COOH group of
Asp and the –OH group of Ser, which
have high polarity, are good covalent
catalysts.
• Serine proteases, such as trypsin,
chymotrypsin and thrombin are some of
the examples of the enzymes acting by
the covalent catalytic mechanism.
• This form of catalysis is also called as
nucleophillic catalysis.

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

27. Proximity catalysis
• The reactants should come in close
proximity to the enzyme for
appropriate catalysis to occur.
• The higher the concentration of the
substrate molecules, the greater will
be the rate of reaction.

28. ENZYME INHIBITION
• Chemical substances which inhibit
enzyme activity and reduce the
velocity of an enzyme catalyzed
reaction are called inhibitors, e.g.
cyanide inhibits cytochrome
oxidase.
• The phenomenon of a decrease in
enzymatic reaction brought about
by the addition of an inhibitor is
called enzyme inhibition.
• There are 3 broad categories of
enzyme inhibition.
1. Reversible inhibition
2. Irreversible inhibition
3. Allosteric inhibition

29. REVERSIBLE INHIBITION
• The inhibitor binds non-covalently
with enzyme.
• The enzyme inhibition can be
reversed if the inhibitor is
removed.
• The reversible inhibition is further
sub-divided into
1. Competitive inhibition
2. Non-competitive inhibition
3. Uncompetitive inhibition

30. Competitive inhibition
• A substance that competes directly with a normal substrate for an enzyme’s
substrate-binding site is known as a competitive inhibitor.
• Chemical structure of the competitive inhibitor closely resembles with that of the
substrate. They are called structural analogs.
• The inhibitor competes with the substrate for binding at the active site of the
enzyme.
• The inhibitor forms a complex with the enzyme called as enzyme-inhibitor complex
(EI), instead of the enzyme substrate complex (ES).
• A competitive inhibitor thus reduces concentration of free enzyme available for the
substrate binding.
• The relative concentration of the substrate and inhibitor and their respective affinity
with the enzyme determines the degree of competitive inhibition.
• The degree of inhibition can be reduced by increasing the concentration of the
substrate.
• In competitive inhibition, the Km value increases whereas Vmax remains unchanged.
• For example, sulphanilamide (a sulfa drug) is an antibacterial agent and resembles p-
aminobenzoic acid (PABA), structurally. The drug is a competitive inhibitor of the
enzyme dihydropteroate synthase, in bacteria.
• Similarly, methotrexate, a structural analog of folate, competitively inhibits
dihydrofolate reductase and is used in the treatment of childhood leukemias.

31. Ethanol in the treatment
of methanol poisonining
• Methanol as such is only mildly
toxic, in the liver.
• when acted upon by the enzyme
alcohol dehydrogenase, methanol
is converted into a highly toxic
compound, i.e. formaldehyde.
• The toxicity of methanol can be
overcome by giving ethanol.
• Ethanol competes with methanol
for binding to the active site of the
enzyme and slows down the
conversion of methanol to
formaldehyde, this in turn
facilitates the excretion of
methanol from the body in the
urine, without being converted
into formaldehyde.

32. Clinical applications of competitive enzyme inhibition
Drug Enzyme True substrate Clinical application
Allopurinol Xanthine oxidase Hypoxanthine Gout
Sulfonamides Dihydropteroate
synthase
Para-amino benzoic
acid (PABA)
Bacterial infection
Methotrexate Dihydrofolate
reductase
Dihydrofolate Cancer
Dicumarol Epoxide reductase Vitamin K epoxide Thrombosis
Ethanol (alcohol) Alcohol
dehydrogenase
Ethanol (alcohol) Methanol poisoning
Succinyl choline Acetyl cholinesterase Acetyl choline Muscle relaxant

33. Non-competitive inhibition (mixed inhibition)
• The inhibitor (I) usually bears no structural similarity to the substrate(S) and
thus there occurs no competition between I and S.
• A non-competitive inhibitor binds at a site other than the substrate binding site
hence the enzyme as well as the enzyme-substrate complex can bind to
inhibitor. Therefore, both binary(EI) and ternary (ESI) complexes can be formed.
• Since ESI may breakdown to form a product but at a slower rate, therefore, this
type of inhibition is also known as mixed inhibition.
• For non-competitive inhibition, the Km value is unchanged whereas Vmax is
lowered.
• Certain analogs of purines and pyrimidines (called antimetabolites) are non-
competitive inhibitors of some of the enzymes and are used as
chemotherapeutic agents, e.g. 5-flurouracil. It is an analog of thymine and
inhibits thymidylate synthetase, noncompetitively.
• Deoxycycline, an antibiotic, functions at low concentrations as a non-
competitive inhibitor of a proteolytic enzyme, collagenase. It is used to treat
periodontal disease.
• Metal ions at lower concentrations act as reversible non-competitive inhibitors.

36. Uncompetitive inhibition
• Uncompetitive inhibition occurs
when the inhibitor binds after the
substrate has bound to the enzyme,
and then stops the reaction.
• Uncompetitive inhibitor can bind
only to the enzyme-substrate(ES)
complex and such an inhibitor may
not resemble the substrate.
• In uncompetitive inhibition both Km
and Vmax are decreased.
• An example is the inhibition of
alkaline phosphatase by
phenylalanine.

37. IRREVERSIBLE INHIBITION
• An irreversible inhibitor bind covalently with the enzymes and inactivate them
which is irreversible.
• Several oxidizing agents, enzyme poisons, and heavy metals cause irreversible
inhibition of enzyme activity.
• These inhibitors bear no structural similarity to the substrate, the inhibition
cannot be reversed by increasing substrate concentration.
• Irreversible inhibitors can be divided in to three categories:
1. Group specific inhibitors
2. Reactive substrate analogs inhibitor or affinity labels
3. Suicide inhibitor resulting or mechanism based inactivation.

38. Group Specific Irreversible Inhibitor
• These inhibitors react with specific R-groups of amino acid residues in the
enzyme that plays essential roles in catalysis, substrate binding or
maintenance of the enzyme’s functional conformation.
• Diisopropylphosphofluoride (DIPF) can inhibit an enzyme acetylcholine
esterase by covalently reacting, with hydroxyl group of an essential serine
residue present at the active site of the enzyme. All DIPF inhibited enzyme
have an essential serine residue in their active site.
• Iodoacetamide which can react with sulfhydryl (-SH) groups of essential
cysteine residues or with the imidazole group of essential histidine residues of
the enzyme.
• Heavy metals like Pb2+, Ag+, Hg2+, and etc. form tight covalent bond with
essential SH-group of cysteine residues of the enzyme.

39. Reactive Substrate Analogs or Affinity Labels
• Reactive substrate analogs are molecules that are structurally similar to the
substrate for an enzyme.
• These substrate analogs possess a highly reactive group which is not present
in the natural substrate.
• The reactive group of affinity labels covalently reacts with amino acid
residues of the active site and permanently blocks the active site of the
enzyme.
• Tosyl-L-phenylalanine chloromethyl ketone (TPCK): It is a reactive substrate
analog of the normal substrate for the enzyme chymotrypsin. TPCK binds at
the active site of the enzyme and then reacts irreversibly with an essential
histidine residues at that site and inhibits the enzyme.
• 3-Bromoacetol phosphate (BAP): It is a reactive substrate analog of the
normal substrate dihydroxyacetone phosphate (DHAP) for the enzyme triose
phosphate isomerase (TIM) of glycolysis. BAP binds at the active site of the
enzyme TIM and covalently modifies a glutamic acid residue required for
enzyme activity.

40. Suicide inhibition
• Suicide inhibition is a specialized
form of irreversible inhibition.
• In 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 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.

41. ALLOSTERIC INHIBITION
• Some of the enzymes possesses additional sites, known as allosteric sites
(Greek: allo- other), besides the active site. Such enzymes are known as
allosteric enzymes or regulatory enzymes.
• The catalytic activity of some of the regulatory enzymes is modulated by
certain low-molecular weight substances.
• Since these substances exhibit their effect after they bind at the site which is
different from the catalytic site, i.e. they occupy another space, hence these
substances are called as allosteric effectors (allosteric modulators).
• The catalytic site where the substrate binds, and the allosteric site
which is occupied by the effector molecule, are physically distinct and
often located far away from each other.
• The effector may activate an enzymatic reaction (allosteric activation)
and is called as a positive effector or allosteric activator.
• The binding of the effector molecule may also result in inhibition of the
enzymatic reaction. This is called as allosteric inhibition and such a
effector molecule is called as a negative effector or allosteric inhibitor.

42. • Reversal of such an inhibition can be brought about by increasing the amount
of the substrate, relative to the amount of the inhibitor.
• If the effector substance is the substrate itself, it is called as the homotropic
effect.
• On the other hand, if the effector molecule is a substance other than the
substrate, then it is called as the heterotropic effect.
• Allosteric enzymes are divided into two classes based on the influence of
allosteric effector on Km and Vmax .
1. K-class of allosteric enzymes
• The effector changes the Km and not the Vmax .
• E.g. phosphofructokinase
2. V-class of allosteric enzymes
• The effector alters the Vmax and notnthe Km .
• E.g. acetyl CoA carboxylase
 Conformational change in the allosteric enzymes
• Most of the allosteric enzymes are oligomeric in nature.
• 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.

44. Allosteric modulation of some enzymes
Enzyme Allosteric activator Allosteric inhibitor
Hexokinase ADP Glucose-6-P, ATP
Isocitrate dehydrogenase ADP Glucose-6-P, ATP
Glutamate dehydrogenase ADP ATP, NADH
Pyruvate carboxylase Acetyl CoA ADP

45. 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.
1. Allosteric regulation
2. Activation of latent enzymes
3. Compartmentation of metabolic pathways
4. Control of enzyme synthesis
5. Enzyme degradation
6. Isoenzymes

46. Feedback regulation
• The process of inhibiting the first
step by the final product in a series
of enzyme catalyzed reactions of a
metabolic pathway is referred to as
feedback regulation.
• When a substrate (A) is converted
to an end product (P) through
various intermediates, such as
B,C,D, etc., the end product of the
reaction inhibits the first enzyme
of the pathway.
• In this type of inhibition,
accumulation of the end product
slows down the whole reaction
sequence. As the end product is
consumed , the synthesis
continues.
• Feedback inhibition or end product
inhibition is a specialized type of
allosteric inhibition.

47. Activation of latent enzymes (covalent modification)
• 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 etc. are
respectively converted to the active enzymes chymotrypsin, pepsin.
• 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.
• This enzyme is active in phosphorylated state.
• There are some enzymes which are active in dephosphorylated state and become
inactive when phosphorylated e.g. glycogen synthase, HMG CoA reductase.
• A few enzymes are active only with sulfhydryl (-SH) groups, e.g. succinate
dehydrogenase, urease.
• Glutathione brings about stability of these enzymes.

49. Compartmentation
• There are certain substances in
the body (e.g., fatty acids,
glycogen) which are synthesized
and also degraded.
• 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.

50. Control of enzyme synthesis
• Most of the enzymes, particularly the rate limiting ones, are present in very
low concentration.
• The amount of the enzyme directly controls the velocity of the reaction,
catalysed by the enzyme.
• Many rate limiting enzymes have short half-lives.
• This helps in the efficient regulation of the enzyme levels.
• There are two types of enzymes
1. Constitutive enzymes (house-keeping enzymes)
• Its levels are not controlled and remain fairly constant.
2. Adaptive enzymes
• Its concentrations increase or decrease as per body needs and are well
regulated.

51. Induction and Repression
• The term induction is used to represent increased synthesis of enzyme while
repression indicates its decreased synthesis.
• Induction or repression determines the enzyme concentration at the gene
level through the mediation of hormones or other substances.
• Example of gene induction: The hormone insulin induces the synthesis of
glycogen synthetase, glucokinase, phosphofructokinase and pyruvate kinase.
• 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.
• If there is sufficient glucose available, there is no necessity for its synthesis.
This is achieved through repression of pyruvate carboxylase by glucose.

52. Enzyme degradation
• Enzymes are not immortal, since it
will create a series of problems.
• There is a lot of variability in the
half-lives of individual enzymes.
• E.g. LDH4 – 5 to 6 days; LDH1 – 8 to
12 hours; amylase – 3 to 5 hours.
• Enzymes when not needed, they
immediately disappear and, as
when required they are quickly
synthesized.
Isoenzymes
• Multiple forms of the same enzyme
will also help in the regulation of
enzyme activity.
• Many of the isoenzymes are tissue-
specific.

53. APPLICATIONS OF ENZYMES
THERAPEUTIC IMPORTANCE
1. Streptokinase : is used in blood clot-dissolution during an acute myocardial
infarction or in deep vein thrombosis.
2. Asparaginase : is used for some types of leukemias based on the rationale
that the tumor cells are asparagine-dependent for their multiplication and
survival.
3. Deoxyribonuclease (DNAse) : is administered by the respiratory route to
clear up the viscid secretions in patients of cystic fibrosis.
4. Serratiopeptidase : used to minimize the edema that accompanies a physical
trauma or an acute inflammation of the skin.
5. Hyaluronidase : Used for hypodermoclysis, to facilitate the subcutaneous
administration of water/electrolyte solutions in patients with hypovolemic
shock where the collapsed veins are difficult to locate.
6. Hemocoagulase: is used as a hemostat.

55. DIAGNOSTIC IMPORTANCE
Measurement of various enzymes in serum has been found to be helpful in
the diagnosis of various diseases.

57. Analytical application reagents (for estimation)
Enzymes use
Glucose oxidase and peroxidase Glucose
Urease Urea
Cholesterol oxidase Cholesterol
Uricase Uric acid
Lipase Triacylglycerols
Luciferase To detect bacterial contamination of
foods
Alkaline phosphatase/horse radish
peroxidase
In the analytical technique ELISA

58. ISOENZYMES
Definition
• The multiple forms of an enzyme catalyzing the same reaction are isoenzymes
or isozymes.
• Isoenzymes may be present in different tissues of the same organism, in
different cell types or subcellular compartments.
• Besides the source, they also differ from each other with respect to their
structure, electrophoretic mobility and immunological properties.
Explanation for the existence of isoenzymes
• The most common mechanism for the formation of isoenzymes involves the
arrangement of subunits, arising from different genetic loci, in different
combinations to form the active polymeric enzyme.
• E.g. malate dehydrogenase of cytosol is different from that found in
mitochondria.
DIAGNOSTIC IMPORTANCE OF ISOENZYMES
• Isoenzymes that have wide clinical applications include lactate
dehydrogenase, creatine phosphokinase and alkaline phosphatase

60. ISOENZYMES OF LACTATE DEHYDROGENASE
• LDH systematic name is L-lactate-NAD+ oxidoreductase (E.C. 1.1.1.27).
• It catalyzes the interconversion of lactate and pyruvate.
 Structure
• Lactate dehydrogenase (LDH) is a tetramer, i.e. it has four polypeptide
subunits.
• Each subunit may be one of the two types, known as the H type (heart-type)
and the M type (muscle-type) 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.
 Diagnostic Significance of LDH
• LDH1 has 4H type of polypeptide chains (H4) and is predominantly found in
myocardium while LDH5 has 4M subunits (M4) and is predominant found in
hepatic tissue.
• The other forms are LDH2 (H3M), LDH3 (H2M2), and LDH4 (HM3).
• LDH1 and LDH2 predominate in myocardium and RBC, while LDH5
predominates in the liver and the skeletal muscle.

61. • LDH1 becomes greater than LDH2
(known as flipped ratio) between 12
and 24 hours following an acute
myocardial infarction.
• Rise in LDH starts 12 to 18 hours
after the onset of acute myocardial
infarction, with peak at 48 hours to
72 hours.
• The levels return to below the upper
normal level after 6 to 10 days.
• Rise in total LDH parallels LDH1
isozyme.
• Marked elevation of the total LDH
activity, upto 50 times the upper
normal value, may be observed in
megaloblastic anemia.
• Elevation of LDH activity is also
observed in liver diseases, renal
diseases and malignancy.
• Normal value of LDH : 100-200 U/L

62. ISOENZYMES OF CREATINE PHOSPHOKINASE
• Creatine kinase (CK) or creatine phosphokinase (CPK) catalyses the inter-conversion
of phosphocreatine (creatine phosphate) to creatine.
• It exists in three forms.
• Each isoenzyme is a dimer composed of two subunits, i.e. M (muscle) type and B
(brain) type.
• The three isoenzymes are CPK1 (BB) found in brain, CPK2 (MB) in myocardium, and
CPK3 (MM) in skeletal muscle.
• CPK-MB isoenzyme is present in very small amount in serum i.e. almost
undetectable in serum with less than 2% of total CPK.
• CK activity is greatly elevated in all types of muscular dystrophies. Usually only CK-3
is present in serum, in case of dystrophies and myopathies.
• CPK2 isoenzyme is not elevated in skeletal muscle disorders . Therefore , estimation
of the enzyme CPK2 (MB) is the earliest reliable indication of myocardial infarction.
• Its level rises within 4-6 hours in acute myocardial infarction and reaches to a
maximum within 1 day of the infarction while return to normal occurs within 48-72
hours.
• Serum CK-1 activity may increase in patients with head injury.
• CK activity is greatly elevated in all types of muscular dystrophies.
• Normal value: Male : 15-100 U/L
Female : 10 – 80 U/L

63. ISOENZYMES OF ALKALINE PHOSPHATASE
• Different tissues contain different forms of alkaline phosphatase.
• A major portion of alkaline phosphatase in serum is derived from liver and its level
rises in post-hepatic jaundice.
• As many as six isoenzymes of alkaline phosphatase (ALP) have been identified.
• ALP is a monomer, the isoenzymes are due to the difference in the carbohydrate
content (sialic acid residues).
• The most important ALP isoenzymes are alpha1-ALP, alpha2-heat labile ALP, alpha2
heat stable-ALP, prebeta ALP, Gamma-ALP etc.
• In growing children, the major isoenzyme is from the bone which is related to its
increased osteoblastic activity.
• During the last trimester of pregnancy, there is an increase in alkaline phosphatase
which is of placental origin.
• Estimation of serum alkaline phosphatase (ALP) is of great significance in the
diagnosis of hepatobiliary diseases and bone diseases associated with an increased
osteoblastic activity.
• Among the bone diseases, highest levels are seen in Paget’s disease.
• Only moderate rise is observed in osteomalacia.
• Increase in alpha2-heat labile ALP suggests hepatitis whereas pre beta-ALP indicates
bone diseases.
• Normak value: 40-125 U/L

64. ISOENZYMES OF ALCOHOL DEHYDROGENASE
• Alcohol dehydrogenase (ADH) has two heterodimer isoenzymes.
• Among the white Americans and Europeans, alpha-beta-1 isoenzyme is
predominant whereas in Japanese and chinese (Orientals) alpha-beta-2 is
mostly present.
• The alpha-beta-2 more rapidly converts alcohol to acetaldehyde.
• Accumulation of acetaldehyde is associated with tachycardia and facial
flushing among Orientals which is not commonly seen in whites.
• It is believed that Japanese and Chinese have increased sensitivity to alcohol
due to the presence of alpha-beta-2 isoenzyme of ADH.
• Normal value : 0.07 – 0.56 U/L

65. ENZYME PATTERN IN DISEASES
ENZYMES IN MYOCARDIAL INFARCTION

67. ENZYMES PATTERN IN OTHER DISEASES

68. ENZYME KINETICS AND Km VALUE
• The study of reaction rates and how they change in response to changes in
experimental parameters is known as kinetics.
• The relationship between substrate concentration and reaction velocity can
be derived by Michaelis-Menten equation.
• Michaelis and Menten proposed that an enzyme (E) forms enzyme-substrate
complex (ES), with a single substrate (S).
• The complex (ES) is broken down, relatively slowly, into free enzyme (E) and
the product (P).
K1 K3
• E + S --------------> E--S ---------------> E + P
K2
If concentration of substrate is increased, the forward reaction K1 is increased,
and so K3 as well as total velocity is correspondingly enhanced. The three
different constants may be made into one equation,

69. Km = K2 + K3
K1
Km is called as Michaelis Constant.
It is further shown that
Velocity (v) = Vmax [S]
Km + [S]
When concentration of substrate is made equal to Km,
i.e.
When [S] = Km
Velocity (v) = Vmax [S] = Vmax [S] = Vmax
[S] + [S] 2 [S] 2
• or v = ½ Vmax

70. MICHAELIS-MENTEN CONSTANT(Km)
• It is defined as the substrate concentration (expressed in moles/l) to produce
half-maximum velocity in an enzyme catalyzed reaction.
• It indicates that half of the enzyme molecules (i.e. 50%) are bound with the
substrate molecules when the substrate cocentration equals the Km value.
Salient Features of Km
1. Km value is substrate concentration (expressed in moles/L) at half-maximal
velocity.
2. It denotes that 50% of enzyme molecules are bound with substrate molecules
at that particular substrate concentration.
3. Km is independent of enzyme concentration. If enzyme concentration is
doubled, the Vmax will be double. But the Km will remain exactly same. In other
words, irrespective of enzyme concentration, 50% molecules are bound to
substrate at that particular substrate concentration.

71. 4. Km is the Signature of the Enzyme. Km value is thus a constant for an enzyme.
It is the characteristic feature of a particular enzyme for a specific substrate.
5. The affinity of an enzyme towards its substrate is inversely related to the
dissociation constant, Kd for the enzyme–substrate complex.
K1 K3
• E + S -----→ E–S complex ---------→ E + P
K2
• Kd = K1 and Km= K2 + K3
K2 K1
Therefore, the smaller the tendency for the dissociation of the complex, the
greater is the affinity of the enzyme for the substrate.
6. Km denotes the affinity of enzyme for substrate. The lesser the numerical
value of Km, the affinity of the enzyme for the substrate is more.
7. For majority of the enzymes, the Km value are in the range of 10-5 to 10-2 .

72. SIGNIFICANCE OF Km
1. Physiological significance
• Glucose can be phosphorylated to glucose 6-phosphate by glucokinase (the
enzyme present in the liver, is specific for glucose) or hexokinase
(nonspecific for hexoses) except galactose, present in all tissues).
Glucokinase has a high Km (i.e. low affinity for glucose), hence it is important
during the fed state when glucose is in excess. On the other hand, under
post-absorptive/fasting conditions, hexokinase having a low Km (i.e. high
affinity for glucose) is important so that glycolysis continues to provide
energy to the vital organs even at low blood glucose levels.
2. Laboratory significance
• During enzyme assay in the laboratory, the substrate concentration is kept at
saturating amounts (at least 10 times the Km) so that the reaction proceeds to
completion.

73. 3. Clinical significance
• The Km value for a given enzyme may differ person to person and explains the
varied response to drugs/chemicals.
• Aldehyde dehydrogenase enzyme oxidizes acetaldehyde (formed from alcohol)
into acetic acid. People having a low Km (i.e. high affinity) variant of the
enzyme metabolize acetaldehyde rapidly and are less susceptible to its
adverse effects such as headche and flushing. On the other hand, having the
high Km variant are more prone to these side effects of alcohol.

74. LINEWEAVER-BURK DOUBLE RECIPROCAL PLOT
• Sometimes it is impractical to achieve high substrate concentrations to reach
the maximal velocity conditions. So, ½ Vmax or Km may be difficult to
determine.
• Then, the experimental data at lower concentrations is plotted as reciprocals.
• The straight line thus obtained is extra plotted to get the reciprocal of Km .This
is called Lineweaver-Burk Plot or Double Reciprocal Plot which can be derived
from the Michaelis-Menten equation.
v = Vmax [S]
Km + [S]
When inverted, the equation is:
1 = Km + [S]
v Vmax [S]
= Km × 1 + [S]
Vmax [S] Vmax [S]
= Km × 1 + 1
Vmax [S] Vmax

75. • If we plot 1/V against 1/[S], it will give a straight line graph. Intercepts in X-
axis is minus 1/Km, from which Km can be calculated.
Fig. Lineweaver-Burk plot