Enzymes Classification

1. ENZYMES
Mrs. Prajakta B. Kothawade
Assistant Professor,
PES Modern College of Pharmacy, for ladies, Moshi, Pune

2. • 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.
• Definition of Enzymes:
• 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.

3. • Enzymes are protein in nature, colloidal and thermolabile in
character, and specific in action.
• 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.

4. • In the early days, the enzymes were given names by their
discoverers in an arbitrary manner. For example, the names
pepsin, trypsin and chymotrypsin convey no information about
the function of the enzyme or the nature of the substrate on
which they act.
• Sometimes, the suffix-ase was added to the substrate for
naming the enzymes e.g. lipase acts on lipids; nuclease on
nucleic acids; lactase on lactose. These are known as trivial
names of the enzymes

5. Enzymes are sometimes considered under two broad categories :
(a) Intracellular enzymes - They are functional within cells where they are
synthesized.
(b) Extracellular enzymes - These enzymes are active outside the cell; all the
digestive enzymes belong to this group.
The International Union of Biochemistry (lUB) appointed an Enzyme
Commission in 1961.
Since 1964, the IUB system of enzyme classification has been in force.
Enzymes are divided into six major classes (in that order).
Each class on its own represents the general type of reaction brought about by the
enzymes of that class.

6. 1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases or Desmolases
5. Isomerases
6. Ligases or Synthetases

8. 1. Oxidoreductases:
• Enzymes which bring about oxidation-reduction
reactions between two substrates.
• Groups to be present in the substrate: CH—OH, C=O,
CH—CH, CH—NH2 and CH=NH groups
• Eg., Alcohol dehydrogenase, Acyl-CoA dehydrogenase,
Cytochrome oxidase etc.,

9. 2. Transferases:
• Enzymes which catalyze the transfer of a group, G
(other than hydrogen) between a pair of substrates, S
and S′ are called transferases
S—G + S′ -----------→ S + S′—G
• These enzymes catalyze the transfer of one-carbon
groups, aldehydic or ketonic residues and acyl,
glycosyl, alkyl, phosphorus or sulfur-containing groups
• Eg., Acyltransferases, Glycosyltransferases, Hexokinase

10. 3. Hydrolases:
• These catalyze the hydrolysis of their substrates by
adding constituents of water across the bond they
split.
• The substrates include ester, glycosyl, ether, peptide,
acid-anhydride, C—C, halide and P—N bonds.
• e.g., glucose-6-phosphatase, pepsin, trypsin,
esterases, glycoside hydrolases

11. 4. Lyases (Desmolases):
• These are those enzymes which catalyze the
removal of groups from substrates by mechanisms
other than hydrolysis, leaving double bonds.
• These include enzymes acting on C—C, C—O, C—N,
C—S and C—halide bonds.
• Eg., Aldolase, Fumarase, Histidase etc.,

12. 5. Isomerases:
1. These catalyze interconversion of optical, geometric
or positional isomers by intramolecular
rearrangement of atoms or groups.
2. Eg., Alanine racemase, Retinene isomerase,
Glucosephosphate isomerase etc.,

13. 6. Ligases:
• These are the enzymes catalyzing the linking together
of two compounds utilizing the energy made available
due to simultaneous breaking of a pyrophosphate
bond in ATP or a similar compound.
• This category includes enzymes catalyzing reactions
forming C—O, C—S, C—N and C—C bonds.
• Eg., Acetyl-CoA synthetase, Glutamine synthetase etc.,

14. The functional unit of the enzyme is known as holoenzyme which
is often made up of apoenzyme (the protein part) and a coenzyme
(non-protein organic part).
Holoenzyme Apoenzyme + Coenzyme
(active enzyme) (protein part) (non-protein part)
CHEMICAL NATURE OF ENZYMES

15. • Monomeric enzyme : is used if it is made up of a single
polypeptide e.g. ribonuclease, trypsin.
• Oligomeric enzymes: Some of the enzymes which possess
more than one polypeptide (subunit) chain. e.g. lactate
dehydrogenase, aspartate transcarbamoylase etc.
• Multienzyme complexes: possessing specific sites to
catalyze different reactions in a sequence. Only the native
intact multienzyme complex is functionally active and not the
individual units, if they are separated e.g. pyruvate
dehydrogenase, fatty acid synthase, prostaglandin synthase
etc..

16. Specificity of enzymes
• Enzymes are highly specific in their action
• Specificity is a characteristic property of the active site
• Types of enzyme specificity:
• Stereospecificity
• Reaction specificity
• Substrate specificity

17. 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
• L-amino acid oxidase and D-amino acid oxidase act on L- and D-
amino acids respectively.

18. • 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
stereospecificity, since they are specialized in the
interconversion of isomers

19. 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.

20. Substrate specificity
• 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
• Relative substrate specificity:
• Some enzymes act on structurally related substances,
• May be dependent on the specific group or a bond present.
• The action of trypsin is a good example for group specificity

21. • Bond Specificity:
• Most of the proteolytic enzymes are showing group (bond)
specificity.
• E.g. trypsin can hydrolyse peptide bonds formed by carboxyl
groups of arginine or lysine residues in any proteins
• Group Specificity:
• One enzyme can catalyse the same reaction on a group of
structurally similar compounds,
• E.g. hexokinase can catalyse phosphorylation of glucose,
galactose and mannose.

22. COENZYMES
• The non-protein, organic, Iow molecularweight
and dialysable substance associated with
enzyme function is known as coenzyme.
• Coenzymes are often regarded as the second
substrates or co-substrates, since they have
affinity with the enzyme comparable with that
of the substrate
• Types of coenzymes: B-complex vitamin
coenzymes and non B-complex vitamin
coenzymes

25. COFACTORS
• The non-protein, inorganic, Iow molecular weight
and dialysable substance associated with enzyme
function is known as cofactors.
• Most of the cofactors are metal ions
• Metal activated enzymes: In these enzymes, the
metals form a loose and easily dissociable
complex.
• Eg., ATPase (Mg2+ and Ca2+, Enolase (Mg2+)

26. • Metalloenzymes: In this case metal ion is bound
tightly to the enzyme and is not dissociated
Eg., alcohol dehydrogenase, carbonic anhydrase,
alkaline phosphatase, carboxypeptidase and aldolase
contain zinc.
Phenol oxidase (copper)
Pyruvate oxidase (manganese)
Xanthine oxidase (molybdenum)
Cytochrome oxidase (iron and copper).

27. ACTIVE SITE
The active site (or active center) of an
enzyme represents as the small region
at which the substrate binds and
participates in the catalysis
Salient features:
• The existence of active site is due
to the tertiary structure of protein.
• Made up of amino acids which are
far from each other in the linear
sequence of amino acids.

28. • Active sites are regarded as clefts or crevices or
pockets occupying a small region in a big enzyme
molecule.
• The active site is not rigid, it is flexible to promote the
specific substrate binding
• Enzymes are specific in their function due to the
existence of active sites.

29. • Active site possesses a substrate binding site and a
catalytic site.
• The coenzymes or cofactors on which some
enzymes depend are present as a part of the
catalytic site.
• The substrate binds at the active site by weak
noncovalent bonds.

30. • The commonly found amino acids at the active
sites are serine(mostly found), aspartate, histidine,
cysteine, lysine, arginine, glutamate, tyrosine .
• The substrate 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.

31. MODE OF ENZYME ACTION
• Two theories have been put forth to explain
mechanism of enzyme-substrate complex formation
1. Lock and key model/ Fischer’s template Theory
2. Induced fit theory/Koshland’s model

32. Lock and key model/ Fischer’s template Theory:
• Proposed by a Emil Fischer.
• Very first model proposed to explain an enzyme
catalyzed reaction
• According to this model, the structure or conformation
of the enzyme is rigid.
• The substrate fits to the binding 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.

33. • This model was not accepted because
1. Does not give any scope for the flexible nature of
enzymes
2. Totally fails to explain many facts of enzymatic
reactions
3. Does not explain the effect of allosteric modulator

34. 2. Induced fit theory/Koshland’s model:
• Koshland proposed this model
• The active site is not rigid and pre-shaped
• The interaction of the substrate with the enzyme
induces a fit or a conformation changei n the enzyme,
resulting in the formation of a strong substrate
binding site.
• Further more the appropriate amino acids of the
enzyme are repositioned to form the active site and
bring about the catalysis

35. • This model was accepted because:
1. Has sufficient experimental evidence from the X-ray
diffraction studies.
2. This model also explains the action of allosteric
modulators and competitive inhibition on enzymes

36. FACTORS AFFECTING ENZYME
ACTION
Concentration of the enzyme:
• As the concentration of the enzyme is increased, the
velocity of the reaction proportionately increases.
• This property of enzyme is made use in determining
the serum enzymes for the diagnosis of diseases.

38. Concentration of the Substrate:
• Increase in the substrate concentration gradually
increases the velocity of enzyme reaction within the
limited range of substrate levels.
• A rectangular hyperbola is obtained when velocity is
plotted against the substrate concentration.
• Three distinct phases of the reaction are observed in
the graph (A-linear; B-curve; C-almost unchanged).

39. Order of reaction :
• When the velocity of the reaction is almost
proportional to the substrate concentration, the rate
of the reaction is said to be first order with respect
to substrate.
• When the substrate concentration is much greater
than Concentration of enzyme, the rate of reaction is
independent of substrate concentration, and the
reaction is said to be zero order.

41. 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.

42. • Temperature coefficient or Q10 is defined as increase
in enzyme velocity when the temperature is increased
by 10oC.
• For a majority of enzymes, Q10 is 2 between 0"C and
40oC.
• optimum temperature - 40oC-45oC. (However, a few
enzymes e.g. venom phosphokinases, muscle
adenylatek inase are active even at 100oC. Some plant
enzymes like urease have optimum activity around
60oC.)

43. 1. when the enzymes are exposed to a temperature
above 50oC, denaturation leading to derangement
in the native (tertiary) structure of the protein and
active site are seen.
2. Majority of the enzymes become inactive at
higher temperature (above 70oC).

44. Effect of pH:
• Each enzyme has an optimum pH at which the
velocity is maximum. Below and above this pH, the
enzyme activity is much lower and at extreme pH,
the enzyme becomes totally inactive

45. • Most of the enzymes of higher organisms show
optimum activity around neutral pH (6-8).
• There are, however, many exceptions like pepsin (1-2),
acid phosphatase (4-5) and alkaline phosphatase(10-
11).
• Enzymes from fungi and plants are most active in
acidic pH (4-6).
• Hydrogen ions influence the enzyme activity by
altering the ionic charges on the amino acids
(particularly at the active site) and substrate.

46. Effect of product concentration
ln the living system, this type of inhibition is generally
prevented by a quick removal of products formed

47. Effect of time:
• Under ideal and optimal conditions (like pH,
temperature etc.), the time required for an enzyme
reaction is less.
• Variations in the time of the reaction are generally
related to the alterations in pH and temperature.
Effect of light and radiation:
• Exposure of enzymes to ultraviolet, beta, gamma and
X-rays inactivates certain enzymes.
• The inactivation is due to the formation of peroxides.
• e.g. UV rays inhibit salivary amylase activity.

48. ENZYME KINETICS/MICHAELIS-MENTEN HYPOTHESIS
• Leonor Michaelis and Maud L. Menten (1913), while
studying the hydrolysis of sucrose catalyzed by the
enzyme invertase, proposed this theory.
• According to this theory
• From the above equation theoretically one can explain
the kinetics of the enzyme reaction, but practically not
• For this reason Micheali and Menten proposed an
equation.

49. • From that equation, these immeasurable quantities
were replaced by those which could be easily measured
experimentally.
• Following symbols may be used for deriving Michaelis-
Menten equation :
(Et) = total concentration of enzyme
(S) = total concentration of substrate
(ES) = concentration of enzyme-substrate
complex
(Et) − (ES) = concentration of free enzyme

50. Derivation of the equation:
• The rate of appearance of products (i.e., velocity, V)
is proportional to the concentration of the enzyme-
substrate complex.
V α ES
V = k (ES) -------------------- (1)
• The maximum reaction rate, Vm will occur at a point
where the total enzyme Et is bound to the substrate.
Vm α Et
Vm = k (Et) ----------------------(2)

51. • Dividing equation (1) by (2,) we get :
V = k (ES)
---------------
Vm = k (Et)
• ------------ (3)
Now coming back to the reversible reaction,
E + S ES,
one can write the equilibrium constant for dissociation of
ES as Km which is equal to :

52. Michaelis-Menten equation

53. Michaelis-Menten plot
This plot is used to determine the Vm and Km value of
the enzyme

54. Determination of Vm and Km value
When V = ½Vm
Km = S

55. Significance of Vm and Km value
• Km or Michaelis-Menten constant is defined as the
substrate concentration (expressed in moles/l) to
produce half-maximum velocity in an enzyme catalyzed
reaction
• The Km values of the enzymes differ greatly from one
to other, but it is a characteristic feature of a particular
enzyme.
• for most of the enzymes, the general range is between
10−1 and 10−6M

56. • The Km value depends on the particular substrate and
on the environmental conditions such as temperature
and ionic concentration.
• But it is not dependent on the concentration of enzyme
• Km is a measure of the strength of ES complex. The high
Km value indicates weak binding whereas the low Km
value signifies strong binding.
• The maximal rate (Vm) represents the turnover number
of an enzyme, if the concentration of the active sites
(Et) is known.

57. 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.
• Inhibitor may be organic or inorganic in nature.
• There are three broad categories of enzyme inhibition
1. Reversible inhibition.
2. Irreversible inhibition.

58. 1. Reversible inhibition:
• The inhibitor binds non-covalently with enzyme
• Enzyme inhibition can be reversed if the inhibitor is
removed.
• The reversible inhibition is further sub-divided into
l. Competitive inhibition
ll. Non-competitive inhibition

59. l. Competitive inhibition
• The rate of inhibition depends on:
1. Concentration of substrate and inhibitor
2. Affinity of inhibitor towards the enzyme
• The inhibition can be reversed by increasing the
concentration of substrate

60. • Km value increases whereas Vmax remains
unchanged
• Eg., succinate dehydrogenase
Original substrate - succinic acid
Inhibitor – malonic acid, glutaric acid, oxalic acid
• Competitive inhibitors have clinical significance

62. ll. Non-competitive inhibition
• The rate of inhibition depends on the concentration
of the inhibitor
• Km remains constant whereas Vmax value
decreases

63. • Eg., Various heavy metals ions (Ag+, Hg2+, Pb2+) inhibit
the activity of a variety of enzymes.
• Urease, for example, is highly sensitive to any of these
ions in traces.
• Heavy metals form mercaptides with sulfhydryl (-SH)
groups of enzymes:
• cyanide and hydrogen sulfide strongly inhibit the action
of iron-containing enzymes like catalase and
peroxidase.

64. 2. Irreversible inhibition:
• The inhibitors bind covalently with the enzymes and
inactivate them irreversibly
• These inhibitors are usually toxic poisonous
substances
• Irreversible inhibitors combine with or destroy a
functional group on the enzyme that is essential for
its activity

65. • Eg., lodoacetate – irreversible inhibitor of papain and
glyceraldehyde 3-phosphate dehydrogenase .
Iodoacetate combines with sulfhydryl (-SH) groups at
the active site of these enzvmes and makes them
inactive
• Eg., 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

66. • Eg., 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
• Eg., Penicillin antibiotics act as irreversible inhibitors
of serine – containing enzymes, and block the
bacterial cell wall synthesis

67. ENZYME REGULATION
Covalent modification:
• 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
1. phosphorylation and dephosphorylation
2. oxidation and reduction of disulfide bonds.

68. Covalent modification by phosphorylation-
dephosphorylation of a seryl residue
For some enzymes phosphorylation increases its
activity whereas for some other enzymes it decreases
the activity

70. Covalent modification by oxidation and reduction of
disulfide bonds
• A few enzymes are active only with sulfhydryl (-SH)
groups, Eg., succinate dehydrogenase, urease.
• Substances like glutathione bring about the stability
of these enzymes.

71. Allosteric regulation:
• They possess sites called allosteric site (other than that
of active site)
• Certain substances referred to as allosteric modulators
(effectors or modifiers) bind at the allosteric site and
regulate the enzyme activity.
• positive (+) allosteric effector – the binding of which
increases the activity of the enzyme – so called as
allosteric activator
• negative (-) allosteric effector – the binding of which
decreases the activity of the enzyme – so called as
allosteric inhibitor

72. Homotropic effect: modulator and substrate are same –
mostly positive
Heterotropic effect: modulator and substrate are
different – may be positive or negative

73. Allosteric regulation mechanism