Notes on Enzyme

16/07/08 Enzyme Pharmaceutical
DHN CHANDAN – Department of ACCT, RU (Mobile – 88-01713413431) 1
1. What is enzyme?
Enzyme: Enzyme may be defined as a soluble colloidal biocatalyst which is produced by living cells,
protein in nature (exception – RNA acting as ribozyme), specific in action, capable of catalyzing a
chemical reaction without being altered or destroyed at the end of the process and does not change
the equilibrium constant of the reaction but increases the rate at which the reaction approaches
equilibrium. Or,
Enzyme may be defined as a reaction specific, thermo-labile, non-dialyzable protein catalyst,
produced by the living cells, capable of catalyzing a bio-chemical reaction and reverts to its original
state when the reaction is over.
Most of the enzymes are soluble in water. These are inactive at 0℃ and destroyed by heating at
100℃. Enzymes promote and control the conversion of the complex carbohydrates, proteins, fats into
simple substances which intestine can absorb. They are usually colorless but some are yellow, blue,
green etc. the very existence of life is unimaginable without the presence of enzymes.
2. What is the nature of enzyme? On which factors stability of enzyme depends?
Nature: Enzymes are protein in nature. So they can be inactivated or destroyed by excessive heat
and are precipitated by salts of heavy metals.
Factors: Following are the factors on which enzyme activity depends:
i. 𝑝𝐻
ii. Temperature and
iii. Concentration of a solution.
3. Why enzymes are not dialyzable?
Enzymes are made of protein. As proteins cannot be dialyzed, so enzymes are not dialyzable.
4. Tell the division of enzyme.
Chemically enzymes are divided into two groups:
a) Simple protein enzyme: They consist of simple protein.
b) Complex protein enzyme or conjugate enzyme: They consist of a protein part, apoenzyme
and a prosthetic group, coenzyme. Combination of apoenzyme with coenzyme constitutes the
holoenzyme. Thus holoenzyme is conjugate enzyme.
5. How can you classify enzymes?
Principally enzymes are three classes. They are:
A. Coenzyme: These are the enzymes that required some non-protein compounds known as
co-factor in order to perform their catalytic activity.
B. Apoenzyme: When enzymes do not possess the co-factor, the remaining conjugated
proteins are called apoenzymes.
C. Holoenzyme: When enzymes possess chemical groups that are non-amino acid in nature,
these conjugated proteins are called holoenzymes.
Enzymes are sometimes considered under two broad categories. They are:
A. Intracellular enzymes: They are functional within cells where they are synthesized.
B. Extracellular enzymes: These enzymes are active outside the cell (e.g. digestive enzymes).

2 DHN CHANDAN – Department of ACCT, RU (Mobile – 88-01713413431)
The International Commission of Enzyme in 1961 has recommended a systematic method of
nomenclature and classification of enzymes. According to this system enzymes are divided into six
main groups depending upon their chemical reaction type and reaction mechanism. They are:
A. Oxidoreductases:
Reaction catalyzed: Catalyze oxidation-reduction reaction between two substrates. The
hydrogen donor regarded as the substrate.
𝐴𝐻2 + 𝐵 → 𝐴 + 𝐵𝐻2
Examples: Alcohol dehydrogenase, cytochrome oxidase, L-amino acid oxidase, R-amino acid
oxidase, lactate dehydrogenase etc.
Specific reactions:
i. Lactate dehydrogenase oxidizes lactic acid to pyruvic acid. This enzyme is anaerobic
dehydrogenase as it uses some other substance as hydrogen acceptor.
𝐶𝐻3 − 𝐶𝐻 − 𝐶𝑂𝑂−
|
𝑂𝐻
+ 𝑁𝐴𝐷+
𝒍𝒂𝒄𝒕𝒂𝒕𝒆 𝒅𝒆𝒉𝒚𝒅𝒓𝒐𝒈𝒆𝒏𝒂𝒔𝒆
→
𝐶𝐻3 − 𝐶 − 𝐶𝑂𝑂−
||
𝑂
+ 𝑁𝐴𝐷𝐻 + 𝐻+
Lactate ion Pyruvate ion
ii. Alcohol dehydrogenase oxidizes alcohol to aldehyde or ketone.
Alcohol + 𝑁𝐴𝐷+
𝒂𝒍𝒄𝒐𝒉𝒐𝒍 𝒅𝒆𝒉𝒚𝒅𝒓𝒐𝒈𝒆𝒏𝒂𝒔𝒆
→ Aldehyde / Ketone + 𝑁𝐴𝐷𝐻 + 𝐻+
B. Transferases:
Reaction catalyzed: Catalyze the transfer of some group or radical containing 𝐶, 𝑁 and 𝑃
(other than hydrogen).
𝐴-𝑋 + 𝐵 → 𝐴 + 𝐵-𝑋
Examples: Hexokinase, transaminases, transmethylases, phosphorylase, choline acyl
transferase etc.
Specific reactions:
i. Aminotransferase catalyzes the transfer of nitrogen containing group.
𝐶𝑂𝑂−
|
𝐶 = 𝑂
|
𝐶𝐻2
|
𝐶𝐻2
|
𝐶𝑂𝑂−
+
𝐶𝑂𝑂−
|
𝐻3 𝑁+
− 𝐶 − 𝐻
|
𝑅
𝒂𝒎𝒊𝒏𝒐𝒕𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒂𝒔𝒆
→
𝐶𝑂𝑂−
|
𝐻3 𝑁+
− 𝐶 − 𝐻
|
𝐶𝐻2
|
𝐶𝐻2
|
𝐶𝑂𝑂−
+
𝐶𝑂𝑂−
|
𝐶 = 𝑂
|
𝑅
α-ketoglutaric acid glutamate α-keto acid
ii. Serine hydroxyl-methyl transferase catalyzes the transfer of carbon.
| |
𝑂𝐻 𝑁𝐻3
+
+ 𝑇𝐻𝐹
𝒔𝒆𝒓𝒊𝒏𝒆 𝒉𝒚𝒅𝒓𝒐𝒙𝒚−𝒎𝒆𝒕𝒉𝒚𝒍 𝒕𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒂𝒔𝒆
→
𝐶𝐻2 − 𝐶𝑂𝑂−
|
𝑁𝐻3
+
+
𝑇𝐻𝐹
/
𝐶𝐻2
Serine Glysine
iii. Choline acyl transferase catalyzes the transfer of acyl-containing group.
acetyl-𝐶𝑜𝐴 + choline
𝒄𝒉𝒐𝒍𝒊𝒏𝒆 𝒂𝒄𝒆𝒕𝒚𝒍 𝒕𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒂𝒔𝒆
→ 𝐶𝑜𝐴 + 𝑂-acetyl choline
iv. Hexokinase catalyzes transfer of phosphorous containing group.
𝐴𝑇𝑃 + 𝐷-hexose
𝒉𝒆𝒙𝒐𝒌𝒊𝒏𝒂𝒔𝒆
→ 𝐴𝐷𝑃 + 𝐷-hexose-6-phosphate
C. Hydrolases:
Reaction catalyzed: Catalyze cleavage of bonds (e.g. ester, ether, peptide, glycosyl, acid
anhydride etc.) by addition of water.

𝐴-𝐵 + 𝐻2 𝑂 → 𝐴𝐻 + 𝐵𝑂𝐻
Examples: Lipase, choline esterase, acid and alkaline phosphates, pepsin, urease, rennin,
chymotrypsin, acyl choline acylhydrolase etc.
Specific reactions:
i. Urease acts on urea.
𝐻2 𝑁 − 𝐶𝑂 − 𝑁𝐻2 + 𝐻2 𝑂
𝒖𝒓𝒆𝒂𝒔𝒆
→ 𝐶𝑂2 + 2𝑁𝐻3
ii. β-galactosidase acts on glycosyl compound.
𝛽-𝐷-galactoside + 𝐻2 𝑂
𝜷−𝒈𝒂𝒍𝒂𝒄𝒕𝒐𝒔𝒊𝒅𝒂𝒔𝒆
→ an alcohol + 𝐷-galactose
iii. Acyl choline acylhydrolase acts on exter bonds.
an acylcholine + 𝐻2 𝑂
𝒂𝒄𝒚𝒍 𝒄𝒉𝒐𝒍𝒊𝒏𝒆 𝒂𝒄𝒚𝒍𝒉𝒚𝒅𝒓𝒐𝒍𝒂𝒔𝒆
→ choline + an acid
D. Lyases:
Reaction catalyzed: Catalyze the cleavage of 𝐶 − 𝐶, 𝐶 − 𝑆 and certain 𝐶 − 𝑁 bonds i.e.,
removal of groups ( 𝐻2 𝑂, 𝐶𝑂2, 𝑁𝐻3) from substrates by
mechanisms other than hydrolysis (leaving double bond).
𝐴-𝐵 + 𝑋-𝑌 → 𝐴𝑋-𝐵𝑌
Examples: Aldolase, fumarase, histidase, carbonic anhydrase, pyruvate decarboxylase etc.
Specific reactions:
i. Pyruvate decarboxylase acts on pyruvic acid and splits it into acetaldehyde and 𝐶𝑂2.
𝐻3𝐶 − 𝐶 − 𝐶𝑂𝑂−
||
𝑂
𝒑𝒚𝒓𝒖𝒗𝒂𝒕𝒆 𝒅𝒆𝒄𝒂𝒓𝒃𝒐𝒙𝒚𝒍𝒂𝒔𝒆
→
𝐻3 𝐶 − 𝐶𝐻
||
𝑂
+ 𝐶𝑂2
ii. Carbonic anhydrase acts on carbonic acid and splits it into 𝐻2 𝑂 and 𝐶𝑂2.
𝐻2 𝐶𝑂3
𝒄𝒂𝒓𝒃𝒐𝒏𝒊𝒄 𝒂𝒏𝒉𝒚𝒅𝒓𝒂𝒔𝒆
→ 𝐻2 𝑂 + 𝐶𝑂2
iii. Fumarase catalyze the malate to form fumarate.
𝐿-malate
𝒇𝒖𝒎𝒂𝒓𝒂𝒔𝒆
→ fumerate + 𝐻2 𝑂
E. Isomerases:
Reaction catalyzed: Catalyze interconversion of optical, geometrical or positional isomers
i.e., isomerization reaction.
𝐴 → 𝐴′
Examples: Triose phosphate isomerase, ketoisomerase, retinol isomerase, phosphohexose
isomerase, phosphoglucose isomerase etc.
Specific reactions:
i. Phosphoglucose isomerase converts glucose to fructose.
glucose-6-phosphate
𝒑𝒉𝒐𝒔𝒑𝒉𝒐𝒈𝒍𝒖𝒄𝒐𝒔𝒆 𝒊𝒔𝒐𝒎𝒆𝒓𝒂𝒔𝒆
→ fructose-6-phosphate
ii. Methylmaloxy CoA mulase converts methylmaloxy CoA to succinyl CoA.
𝐶𝐻3
|
𝑂𝑂𝐶 − 𝐶𝐻 − 𝐶 − 𝐶𝑜𝐴−
||
𝑂
𝒎𝒆𝒕𝒉𝒚𝒍𝒎𝒂𝒍𝒐𝒙𝒚 𝑪𝒐𝑨 𝒎𝒖𝒍𝒂𝒔𝒆
⇔
𝑂𝑂𝐶 − 𝐶𝐻2 − 𝐶𝐻2 − 𝐶 − 𝐶𝑜𝐴−
||
𝑂
F. Ligases or synthetases:
Reaction catalyzed: Catalyze formation of bond between 𝐶 − 𝐶, 𝐶 − 𝑆 and 𝐶 − 𝑁 by the
hydrolysis of ATP (coupled to the breaking of a pyrophosphate bond in
ATP or a similar compound).
𝐴 + 𝐵 − − − −⟶ 𝐴-𝐵
ATP ADP + Pi

Examples: Glutamine synthetase, acetyl CoA carboxylase, succinate thiokinase, pyruvate
carboxylase etc.
Specific reactions:
i. Pyruvate carboxylase catalyzes formation of 𝐶 − 𝐶 bond.
𝐻3 𝐶 − 𝐶 − 𝐶𝑂𝑂−
||
𝑂
+ 𝐶𝑂2
𝒑𝒚𝒓𝒖𝒗𝒂𝒕𝒆 𝒄𝒂𝒓𝒃𝒐𝒙𝒚𝒍𝒂𝒔𝒆
→
𝐻𝑂𝑂𝐶 − 𝐶𝐻2 − 𝐶 − 𝐶𝑂𝑂−
||
𝑂
Pyruvate ATP ADP Oxaloacetate
ii. Succinate thiokinase catalyzes formation of 𝐶 − 𝑆 bonds.
𝐺𝑇𝑃 + succinate + 𝐶𝑜𝐴
𝒔𝒖𝒄𝒄𝒊𝒏𝒂𝒕𝒆 𝒕𝒉𝒊𝒐𝒌𝒊𝒏𝒂𝒔𝒆
→ 𝐺𝐷𝑃 + 𝑃𝑖 + succinyl-𝐶𝑜𝐴
iii. Glutamine synthetase catalyzes formation of 𝐶 − 𝑁 bonds.
𝐴𝑇𝑃 + 𝐿-glutamate + 𝑁𝐻4
+ 𝐠𝐥𝐮𝐭𝐚𝐦𝐢𝐧𝐞 𝐬𝐲𝐧𝐭𝐡𝐞𝐭𝐚𝐬𝐞
→ 𝐴𝐷𝑃 + orthophosphate + 𝐿-glutamine
6. Write down the general properties of enzymes.
Following are the properties of enzymes:
a) Active site: Enzyme possesses an active site containing amino acid side chains – interaction
with substrate.
b) Catalytic efficiency: Highly efficient, can increase the rate of reaction up to 103
− 108
times.
One enzyme molecule can react with 100 to 1000 molecules of substrate.
c) Specificity: Highly reaction specific.
d) Co-factors: Some enzymes associate with a non-protein co-factor (known as coenzyme) that is
needed for enzyme activity.
e) Regulation: Enzymes can be inhibited or activated in their function.
 When the product amount is low ⟶ enzymes work more
 When the product amount is high ⟶ enzymes work less
f) Location within the cell: Some are in the cytosol so they can only perform reaction in cytosol,
not in mitochondria or anywhere else.
g) Other properties:
i. Catalyst of living world.
ii. Protein in nature.
iii. Usually globular protein but some are crystalline.
iv. Action is rapid and accurate.
v. Heat labile.
vi. Non-dialyzable.
vii. Colorless (some are yellow, blue, green) and soluble in water, acid solution, salt solution etc.
viii. Precipitates by protein precipitating agents like concentrated alcohol, ammonium sulphate,
trichloroacetic acid etc.
ix. Lowers the activation energy.
x. Don't initiate the reaction.
xi. Huge in size.
xii. Small active complex.
xiii. Effective in very small amount.
xiv. Neither altered / destroyed in the reaction.
xv. Can be activated or inhibited by high temperature, 𝑝𝐻, UV rays, heavy metals etc.

7. What is the active site of the enzymes?
Active site: The active site (or active center) of an enzyme represents as the small region at which
the substrates bind and participate in the catalysis. So, the portion of the enzyme protein molecule
which actually takes part in catalysis is called the active site or catalytic site of the enzyme. If protein
has active site it is enzyme otherwise protein. So, every enzyme is protein but every protein is not
enzyme. Certain common features about the active sites are:
 The active sites are regarded as clefts or crevices or pockets occupying a small volume of the
total protein of an enzyme.
 The active site is a three dimensional activity.
 The specificity of the substrate binding depends upon the arrangement of the atoms or groups
at the active site.
 It is made up of groups that come from the different parts of the linear amino acids chain.
Indeed the residues are far apart in the linear sequence but may come together to bring about
catalysis.
 The active site is not rigid in structure and shape. It is rather flexible to promote the specific
substrate binding.
 Generally, the active site possesses a substrate binding site and a catalytic site. The latter is for
the catalysis of the specific reaction.
 The coenzymes or cofactors on which some enzymes depend are present as a part of the
catalytic site.
 The substrates bind at the active site by weak non-covalent bonds.
 Enzymes are specific in their action due to the existence of active sites.
 The commonly found amino acids at the active sides are serine, aspartate, histidine, cysteine,
lysine, arginine, glutamate, tyrosine etc. Among them serine is most frequently found.
 The substrate [𝑆] binds the enzyme (𝐸) at the active site to form enzyme-substrate complex
(𝐸𝑆). The product (𝑃) is released after the catalysis and the enzyme is available for reuse.
𝐸 + 𝑆 ⇔ 𝐸𝑆 → 𝐸 + 𝑃
8. Describe the mechanism of enzyme action.
The enzyme catalyzed reaction obeys the general laws of chemistry as (i) the reactant (or
substrate) must be colloidal (ii) molecular collision must occur with the correct orientation (iii)
reactant or substrate must have sufficient energy called activation energy. If two reactants colloid with
each other having activation energy, then the reaction takes place.
Figure 1: Energy of activation diagram without and with enzyme

Enzyme reduces the activation energy of substrates. Enzymes can act in several ways to lower 𝛥𝐺.
Such as
 Lowering the activation energy by creating an environment in which the transition state is
stabilized (e.g. straining the shape of a substrate - by binding the transition-state conformation of the
substrate/product molecules, the enzyme distorts the bound substrate (𝑆) into their transition state
form, thereby reducing the amount of energy required to complete the transition).
 Lowering the energy of the transition state, but without distorting the substrate, by creating an
environment with the opposite charge distribution to that of the transition state.
 Providing an alternative pathway. For example, temporarily reacting with the substrate to form
an intermediate 𝐸𝑆 complex, which would be impossible in the absence of the enzyme.
 Reducing the reaction entropy change by bringing substrates together in the correct
orientation to react. Considering 𝛥𝐻 alone overlooks this effect.
There are many hypotheses to explain the mechanism of enzyme action. They are
a. Fisher theory: Emil Fisher in 1894 proposed that the union between the substrate and the
enzyme takes place at the active state more or less in a manner in which a key fits a lock and results in
the formation of an enzyme-substrate complex.
Figure 2: Fisher's lock-key model
b. Michaelis-Menten theory: The enzyme (𝐸) combines with its substrate (𝑆) to form enzyme-
substrate complex (𝐸𝑆), within the complex the substrate breaks down into the product (𝑃) and then
the enzyme dissociates from the substance.
𝐸 + 𝑆 ⇔ 𝐸𝑆 → 𝐸 + 𝑃
c. Koshland’s theory: In 1958 Daniel Koshland suggested a modification to the lock and key
model. An enzyme when occupied by the substrate has a particular geometrical shape. When enzyme
is combined with a substrate the shape of the enzyme molecules altars slightly and because of these
alterations the substrate can become fit properly into the enzyme molecules. In this alter
configuration the enzyme is unstable.
Figure 3: Koshland's induced fit model
9. Write down the characteristics of enzyme actions.
Enzyme action shows the following characteristics:
 Specificity: An enzyme can catalyze one specific reaction by acting only on a particular type of
substrate. For example, amylase acts on starch, lipase acts on lipid etc.

 Enzymes cannot initiate a chemical reaction; they only stimulate or retard the process.
 Enzymes only stimulate or retard the intermediate steps in the chemical process.
 Enzymes are not destroyed in the process, as they are catalytic agent.
 Every enzyme acts best at a limited range of 𝑝𝐻 and shows maximum activity at optimum 𝑝𝐻.
 Every enzyme shows its maximum activity at a particular temperature.
 Enzyme action is generally reversible.
 A small amount of enzyme may act upon unlimited quantity of substrate.
10.Describe the factors that affect the enzymic activity.
The following factors, which influence the rate of enzyme activity, have been summarized below:
A. Substrate Concentration: It has been shown experimentally that if the amount of the enzyme
is kept constant and the substrate concentration is then gradually increased, the reaction velocity will
increase until it reaches a maximum. After this point, increases in substrate concentration will not
increase the velocity (Δ𝐴
Δ𝑇⁄ ). This is represented graphically in Figure.
Figure 4: Effect of substrate concentration
It is theorized that when this maximum velocity had been reached, the entire available enzyme
has been converted to 𝐸𝑆, the enzyme substrate complex. This point on the graph is designated 𝑉𝑚 𝑎𝑥.
Using this maximum velocity
𝐸 + 𝑆
𝐾+1
⟸⟹
𝐾−1
𝐸𝑆
𝐾+2
⟸⟹
𝐾−2
𝑃 + 𝐸
Michaelis developed a set of mathematical expressions to calculate enzyme activity in terms of
reaction speed from measurable laboratory data. The Michaelis constant Km is defined as the
substrate concentration at 1/2 the maximum velocity. This is shown in Figure 8. Using this constant
and the fact that Km can also be defined as:
𝐾 𝑚 =
𝐾+1 + 𝐾+2
𝐾−1
= [𝑆]1
2
𝑉 𝑚𝑎𝑥
𝐾+1, 𝐾−1 and 𝐾+2 being the rate constants from equation (7). Michaelis developed the
following expression for the reaction velocity in terms of this constant and the substrate
concentration.
𝑉 =
𝑉𝑚 𝑎𝑥[𝑆]
𝐾 𝑚 + [𝑆]
Where,

𝑉 = The velocity at any time
[𝑆] = The substrate concentration at this time
𝑉𝑚 𝑎𝑥 = The highest under this set of experimental conditions (𝑝𝐻, temperature etc.)
𝐾 𝑚 = The Michaelis constant for the particular enzyme being investigated
Michaelis constants have been determined for many of the commonly used enzymes. The size
of Km tells us several things about a particular enzyme.
 A small Km indicates that the enzyme requires only a small amount of substrate to become
saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations.
 A large Km indicates the need for high substrate concentrations to achieve maximum
reaction velocity.
 The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently
assumed to be enzyme's natural substrate, though this is not true for all enzymes.
B. Effect of enzyme concentration: In order to study the effect of increasing the enzyme
concentration upon the reaction rate, the substrate must be present in an excess amount; i.e., the
reaction must be independent of the substrate concentration. Any change in the amount of product
formed over a specified period of time will be dependent upon the level of enzyme present.
Graphically this can be represented as:
Figure 5: Zero order reaction rate is independent of substrate concentration
These reactions are said to be "zero order" because the rates are independent of substrate
concentration and are equal to some constant 𝑘. The formation of product proceeds at a rate which is
linear with time. The addition of more substrate does not serve to increase the rate. In zero order
kinetics, allowing the assay to run for double time results in double the amount of product.
Reaction Orders with Respect to substrate Concentration:
Order Rate equation Comments
Zero Rate = 𝑘 Rate is independent of substrate concentration
First Rate = 𝑘[𝑆] Rate is proportional to the first power of substrate concentration
Second Rate = 𝑘[𝑆][𝑆] = 𝑘[𝑆]2
Rate is proportional to the square of the substrate concentration
Second Rate = 𝑘[𝑆1][𝑆2] Rate is proportional to the first power of each of two reactants
The amount of enzyme present in a reaction is measured by the activity it catalyzes. The
relationship between activity and concentration is affected by many factors such as temperature, pH,
etc. An enzyme assay must be designed so that the observed activity is proportional to the amount of
enzyme present in order that the enzyme concentration is the only limiting factor. It is satisfied only
when the reaction is zero order.
In Figure 6, activity is directly proportional to concentration in the area AB, but not in BC.
Enzyme activity is generally greatest when substrate concentration is unlimiting.

Figure 6: Activity vs concentration
When the concentration of the product of an enzymatic reaction is plotted against time, a
similar curve results, Figure 7.
Figure 7: Reaction rate limited by substrate concentration
Between A and B, the curve represents a zero order reaction; that is, one in which the rate is
constant with time. As substrate is used up, the enzyme's active sites are no longer saturated,
substrate concentration becomes rate limiting, and the reaction becomes first order between Band C.
To measure enzyme activity ideally, the measurements must be made in that portion of the curve
where the reaction is zero order. A reaction is most likely to be zero order initially since substrate
concentration is then highest. To be certain that a reaction is zero order; multiple measurements of
product (or substrate) concentration must be made.
C. Effect on the 𝒑𝑯 of the solution: Enzymes are affected by changes in 𝑝𝐻. The most favorable
𝑝𝐻 value - the point where the enzyme is most active - is known as the optimum 𝑝𝐻 (4 − 7). This is
graphically illustrated in Figure 8.
Figure 8: Effect of pH on reaction rate

Extremely high or low 𝑝𝐻 values generally result in complete loss of activity for most enzymes.
𝑝𝐻 is also a factor in the stability of enzymes. The variation in 𝑝𝐻 may influence the ionization of
different groups in the binding and catalytic sites of the enzyme and also in the substrate. As with
activity, for each enzyme there is also a region of 𝑝𝐻 optimal stability. The optimum 𝑝𝐻 value will vary
greatly from one enzyme to another.
𝑝𝐻 for Optimum Activity:
Enzyme 𝑝𝐻 optimum
Lipase (pancreas) 8.0
Lipase (stomach) 4.0 − 5.0
Lipase (castor oil) 4.7
Pepsin 1.5 − 1.6
Trypsin 7.8 − 8.7
Urease 7.0
Invertase 4.5
Maltase 6.1 − 6.8
Amylase (pancreas) 6.7 − 7.0
Amylase (malt) 4.6 − 5.2
Catalase 7.0
D. Effect of the temperature of the solution: Like most chemical reactions, the rate of an enzyme-
catalyzed reaction increases as the temperature is raised. For each enzyme there is a particular
temperature which is called optimum temperature at which the enzyme activity is maximum and the
activity progressively declined both above and below this temperature. A ten degree Centigrade rise in
temperature will increase the activity of most enzymes by 50 to 100%. Variations in reaction
temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results. In the case
of enzymatic reactions, this is complicated by the fact that many enzymes are adversely affected by
high temperatures. As shown in Figure 13, the reaction rate increases with temperature to a maximum
level, then abruptly declines with further increase of temperature. Because most animal enzymes
rapidly become denatured at temperatures above 40℃, most enzyme determinations are carried out
somewhat below that temperature.
Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage of
enzymes at 5℃ or below is generally the most suitable. Some enzymes lose their activity when frozen.
Figure 9: Effect of temperature on reaction rate
E. Effect of product concentration: The concentration of the end products of an enzymic action
has a retarding effect upon the rate of enzyme action. The explanation of retarding the reaction rate
lays on that a more stable complex of enzyme and reaction product forms than that of enzyme and
substrate.

F. Effect of modifiers: Some of the enzymes require certain activators. Activators increase
enzyme velocity through various mechanisms – combining with the substrate, formation of 𝐸𝑆-metal
complex, direct participation in the reaction and bringing a conformational change in the enzyme.
The facts are (i) presence of inorganic metals e.g. 𝑀𝑔++
, 𝐹𝑒++
, 𝑍𝑛++
, 𝐶𝑢++
, 𝑀𝑛++
etc.
increases the catalytic activity of enzyme, (ii) presence of mercury salt, gold, silver etc. decrease the
catalytic activity of enzyme, (iii) rarely, anions are also needed for enzyme activity e.g. chloride ion
(𝐶𝑙−
) for amylase, (iv) formaldehyde destroys enzymes.
G. Effect of coenzyme or cofactor: Enzymes (excepting the enzymes of GIT), work efficiently in
presence of coenzymes and cofactors e.g. 𝑀𝑔++
, 𝐶𝑎++
etc.
H. Effect of hormones: Many hormones can influence the enzyme activity. For example, glucagon
and epinephrine enhance the production of cyclic 𝐴𝑀𝑃 in the liver cells. The cyclic 𝐴𝑀𝑃 in turn
promotes the conversion of enzyme phosphorylase into its active form.
I. Effect of light and radiation: Enzymes are highly sensitive to short wavelength (high energy)
radiation such as x-ray, UV, beta, gamma rays. They form peroxides which cause oxidation of the
enzyme resulting loss in enzyme activity e.g. UV rays inhibit salivary amylase activity.
J. Effect of inhibitor: Enzyme inhibitors are substances which alter the catalytic action of the
enzyme and consequently slow down, or in some cases, stop catalysis. There are three common types
of enzyme inhibition - competitive, non-competitive and substrate inhibition.
Competitive inhibition : Competitive inhibition occurs when the substrate and a substance
resembling the substrate are both added to the enzyme. A theory called the "lock-key theory" of
enzyme catalysts can beused to explain why inhibition occurs.
Figure 10: Lock-key theory - competitive inhibitor
The lock and key theory utilizes the concept of an "active site." The concept holds that one
particular portion of the enzyme surface has a strong affinity for the substrate. The substrate is held in

such a way that its conversion to the reaction products is more favorable. If we consider the enzyme
as the lock and the substrate the key (Figure 10) - the key is inserted in the lock, is turned, and the
door is opened and the reaction proceeds. However, when an inhibitor which resembles the substrate
is present, it will compete with the substrate for the position in the enzyme lock. When the inhibitor
wins, it gains the lock position but is unable to open the lock. Hence, the observed reaction is slowed
down because some of the available enzyme sites are occupied by the inhibitor. If a dissimilar
substance which does not fit the site is present, the enzyme rejects it, accepts the substrate, and the
reaction proceeds normally.
Non-competitive inhibitor: Non-competitive inhibitors are considered to be substances which
when added to the enzyme alter the enzyme in a way that it cannot accept the substrate.
Figure 11: Non-competitive inhibitor
Substrate inhibition: Substrate inhibition will sometimes occur when excessive amounts of
substrate are present. Figure 12 shows the reaction velocity decreasing after the maximum velocity
has been reached.
Figure 12: Substrate become rate inhibiting
Additional amounts of substrate added to the reaction mixture after this point actually
decreases the reaction rate. This is thought to be due to the fact that there are so many substrate

molecules competing for the active sites on the enzyme surfaces that they block the sites (Figure 13)
and prevent any other substrate molecules from occupying them.
Figure 13: Substrate inhibition
This causes the reaction rate to drop since the entire enzyme present is not being used.
In addition to temperature and pH there are other factors, such as ionic strength, which can affect
the enzymatic reaction. Each of these physical and chemical parameters must be considered and
optimized in order for an enzymatic reaction to be accurate and reproducible.
11.What do you know about specificity of enzyme action?
What do you mean by enzyme specificity?
Except few enzymes they are specific in their action. A specific enzyme can act upon a particular
substrate. E.g. zymase acts upon only glucose to give alcohol and 𝐶𝑂2, lactase acts on milk etc.
Thus the enzymes may exhibit in different types of specificities, which are given below:
a) Reaction specificity,
b) Linkage specificity,
c) Stereospecificity or optical specificity,
d) Substrate specificity and
e) Kinetic specificity.
a. Reaction specificity: The same substrate can undergo different types of reactions, each
catalyzed 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.
𝐻
|
𝑅 − 𝐶 − 𝐶𝑂𝑂𝐻
|
𝑁𝐻2
𝒐𝒙𝒊𝒅𝒂𝒔𝒆
→
𝑂
||
𝐻
|
|
𝑁𝐻2
𝒅𝒆𝒄𝒂𝒓𝒃𝒐𝒙𝒚𝒍𝒂𝒔𝒆𝒆
→ 𝑅 − 𝐶𝐻2 − 𝑁𝐻2 + 𝐶𝑂2

b. Linkage specificity: Some enzymes split particular bonds between amino acids in peptide
fragments which are used to determine amino acid sequence in protein. Thus enzymes are linkage
specific.
c. Stereospecificity or optical specificity: Stereoisomers are the compounds which have the same
molecular formula, but differ in their structural configuration. Some enzymes act only on one isomer
and, therefore, exhibit stereospecificity. The class of enzymes belonging to isomerases does not
exhibit stereospecificity, since they are specialized in the interconversion of isomers.
For example, L-amino acid oxidase, a flavin enzyme will act on a L-isomer only but not on a D-
isomer.
𝐻
|
𝑅 − 𝐶 − 𝐶𝑂𝑂−
|
𝑁𝐻3
+
+ 𝑂2 + 𝐻2 𝑂
𝑳−𝒂𝒎𝒊𝒏𝒐 𝒂𝒄𝒊𝒅 𝒐𝒙𝒊𝒅𝒂𝒔𝒆
→
𝑂
||
𝑅 − 𝐶 − 𝐶𝑂𝑂− + 𝑁𝐻4
+
+ 𝐻2 𝑂2
D-amino acid oxidase acts on D-amino acids.
Hexokinase acts on D-hexoses.
Glucokinase acts on D-glucose.
Amylase acts on α-glycosidic linkages.
Cellulase cleaves β-glycosidic bonds.
Maltase hydrolyses α-glucosides.
Stereospecificity is explained by considering three distinct regions of substrate molecule
specifically binding with three complementary regions on the surface of the enzyme.
Figure 14: Diagrammatic representation of stereospecificity (a', b', c') – three-point attachment of substrate
to the enzyme (a, b, c).
d. Substrate specificity: The substrate specificity varies from enzyme to enzyme. It may be
absolute, relative or broad.
i. Absolute substrate specificity: Certain enzymes act only on one particular substrate. For
example, urease acts only on urea to produce ammonia and 𝐶𝑂2.
𝐻2 𝑁 − 𝐶𝑂 − 𝑁𝐻2 + 𝐻2 𝑂
𝒖𝒓𝒆𝒂𝒔𝒆
→ 𝐶𝑂2 + 2𝑁𝐻3
ii. Relative substrate specificity: Some enzymes act on structurally related substances. This, in
turn, may be dependent on the specific group or a bond present. 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). Examples of bond specificity – glycosidases acting on glycosidic bonds of carbohydrates,
lipases cleaving ester bonds of lipids etc.
iii. Broad substrate specificity: Some enzymes act on closely related substrates which is
commonly known as broad substrate specificity. E.g. hexokinase acts on hexoses (glucose, fructose,
mannose, glucosamine but not on galactose). It is possible that some structural similarity among
the first four compounds makes them a common substrate for the enzyme hexokinase.

glucose
fructose
} + 𝐴𝑇𝑃
→
glucose-6-phosphate
fructose-6-phosphate
} + 𝐴𝐷𝑃
e. Kinetic specificity: Many enzymes exhibit a kinetic specificity. E.g. esterase, although
hydrolyzing all esters but the rate of hydrolysis is different for different esters. The amino group
belongs to an aromatic amino group and for the carboxylic group in one of the dicarboxylic amino acid.
12.Give your understanding about folding / active site formation of enzyme.
When enzyme is dissolved in water, then enzyme is folded. This phenomenon is called folding of
enzyme. Folding of enzyme is caused by three kinds of factor:
 Hydrogen bonds,
 Electrostatic bonds (hydrophilic and hydrophobic) and
 Van der Waals bonds.
Hydrogen bonds: Enzymes are the polymer of amino acids. In an enzyme chain >𝑁𝐻 and >𝐶𝑂
groups work in 𝛼-helix and 𝛽-sheets. In fact side chains of 11 to 20 fundamental amino acids can also
participate in hydrogen bonding. 𝛼-helix and 𝛽-sheets are stabilized by the interaction of hydrogen
bond between >𝑁𝐻 and >𝐶𝑂 groups on adjacent chain. Folding in enzyme can be broken by adding
urea and then enzyme is formed in general sequence.
𝛼-helix 𝛽-sheets
Electrostatic bonds (hydrophilic and hydrophobic): Enzyme contains two parts in its molecule
namely hydrophilic and hydrophobic parts. When an enzyme is taken in water, at this every moment
hydrophilic group is attacked by water molecules and on the other hand the hydrophobic group is
repulsed by water molecules occurring an interaction among them. Just at the very moment the
enzyme molecules are folded.
Van der Waals forces: When molecules are taken into water then these are divided into two parts
namely polar and non-polar parts. Almost all the polar parts remain on the surface except very few
polar parts. Non-polar parts are precipitated into water. Not all the non-polar residues remain inside,

some especially shorter ones (glycine, alanine) are on the surface. The non-polar residues almost
everywhere close-packed in Van der Waals contact with their neighbor bond. Water molecules are
attracted to all polar groups on the surface including main chain and >𝑁𝐻 groups. About 75% of the
amino acid residues are arranged in the form of right handed 𝛼-helix both in crystal and solution.
Mechanism: Resists the approach to each of all the atom not actually bonded when these
approach less than a minimum distance (5.4 × 10−8
𝑐𝑚).
13.What do you mean by enzyme modifiers?
Enzyme modifier: The agent that modifies the catalytic activity of the enzyme are known as
enzyme modifiers. Enzyme modifiers are two types:
 Positive modifiers: The agents which increase the catalytic activity of enzyme are known as
positive modifiers. Examples: inorganic metal ions e.g. 𝐹𝑒++
, 𝑀𝑔++
, 𝑍𝑛++
, 𝐶𝑢++
, 𝑀𝑛++
etc.
 Negative modifiers: The agents which decrease the catalytic activity of enzyme are known as
negative modifiers. Examples: mercury, silver, gold, chloroform etc.
14.What is inhibitor? Enumerate the name of common inhibitor available in the market.
Inhibitor: Inhibitor is defined as any substance that can diminish the velocity of an enzyme
catalyzed reaction. Inhibitor binds with enzyme and brings about a decrease in catalytic activity of that
enzyme. The inhibitor may be organic or inorganic in nature. It blocks enzyme activity by two types
namely 𝐶𝑎-channel blocker and 𝛽-blocker.
Inhibitors are of three types. They are:
 Competitive: These resemble real substrate and compete with the substrate to combine
with the catalytic sites of the enzymes.
𝐸
+𝑆
↔ 𝐸𝑆 ⟶ 𝐸 + 𝑃
↓ +𝐼
𝐸𝐼 ↛ 𝑃
 Non-competitive: These do not compete for the active sites. They combine with either free
enzyme or enzyme-substrate complexes.
𝐸 + 𝑆 ⟺ 𝐸𝑆 ⟶ 𝐸 + 𝑃
+ +
𝐼 𝐼
↕ ↕
𝐸𝐼 + 𝑆 𝐸𝐼𝑆
 Uncompetitive: Not very common. Only combines with enzyme-substrate complex.

The name of common inhibitors available in the market and significance of these inhibitors are as
follows:
Inhibitor Significance of inhibitor
Amlodipine (BEX)
Camlodine Sq)
Used for controlling blood pressure.
Ramipril (Sq) Used for controlling blood pressure.
Tenorin ACI) Used as 𝛽-blocker.
Aminopterin
Amethoptein
Methotrexate
Used in the treatment for leukemia and other cancers.
Succinyl chloride Used in surgery for muscle relaxation in anaesthetized patients.
Ephidrena
Amphetamine
Used for elevating catcholamine levels.
Dicumarol
Hydrazide (INH)
Acts as an anticoagulant.
Used leads to 𝐵6 defficiency.
Allopurinol Used in the treatment of gout.
15.What do you mean by substrate?
Substrate: Substrate means the main chemical compound which undergoes alteration in a
chemical reaction upon which the enzyme exerts its influence.
16.Define apoenzyme, coenzyme, cofactor and holoenzyme.
Apoenzyme: The protein part of a holoenzyme is called apoenzyme. So the enzyme without the co-
factor is called apoenzyme or when enzyme does not process the co-factor, the remaining conjugated
proteins are called the apoenzyme. These types of enzymes are inactive.
Coenzyme: Many enzymes catalyze reactions of their substrates only in the presence of specific
heat-stable, low molecular weight organic molecules called coenzyme. We get a factor or group
without amino acid when we hydrolyze protein. This factor is called coenzyme. The coenzyme e.g.
𝐾+
, 𝐹𝑒3+
etc. activates apoenzyme.
Coenzymes are heat-stable, dialyzable, non-protein organic molecules and the prosthetic groups of
enzymes.
Coenzymes are small organic molecules that transport chemical groups from one enzyme to
another. Some of these chemicals such as riboflavin, thiamine and folic acid are vitamins, this is when
these compounds cannot be made in the body and must be acquired from the diet. The chemical
groups carried include the hydride ion (𝐻−
) carried by 𝑁𝐴𝐷 or 𝑁𝐴𝐷𝑃+
, the acetyl group carried by
coenzyme A, formyl, methenyl or methyl groups carried by folic acid and the methyl group carried by
S-adenosylmethionine.
Figure 15: Space-filling model of the coenzyme NADH

Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to
consider coenzymes to be a special class of substrates, or second substrates, which are common to
many different enzymes. For example, about 700 enzymes are known to use the coenzyme NADH.
Coenzymes are usually regenerated and their concentrations maintained at a steady level inside
the cell: for example, 𝑁𝐴𝐷𝑃𝐻 is regenerated through the pentose phosphate pathway and S-
adenosylmethionine by methionine adenosyltransferase.
Cofactor: Some enzymes do not need any additional components to show full activity. However,
others require non-protein molecules called cofactors to be bound for activity. Cofactors can be either
inorganic (e.g., metal ions and iron-sulfur clusters) or organic compounds, (e.g., flavin and heme).
Organic cofactors can be either prosthetic groups, which are tightly bound to an enzyme or
coenzymes, which are released from the enzyme's active site during the reaction. Coenzymes include
𝑁𝐴𝐷𝐻, 𝑁𝐴𝐷𝑃𝐻 and 𝐴𝑇𝑃. These molecules act to transfer chemical groups between enzymes.
Figure 16: Ribbon-diagram showing carbonic anhydrase II. The grey sphere is the zinc cofactor in the active
site
An example of an enzyme that contains a cofactor is carbonic anhydrase and is shown in the ribbon
diagram above with a zinc cofactor bound as part of its active site. These tightly-bound molecules are
usually found in the active site and are involved in catalysis. For example, flavin and heme cofactors
are often involved in redox reactions.
Holoenzyme: Inactive apoenzymes are activated by the cofactor; this activated enzyme is called
holoenzyme. In a word catalytically active enzyme with its cofactor is called holoenzyme. The term
holoenzym can also be applied to enzymes that contain multiple protein subunits, such as the DNA
polymerases; here the holoenzyme is the complete complex containing all the subunits needed for
activity.
Relations: Enzymes that require a cofactor but do not have one bound are called apoenzymes or
apoproteins. An apoenzyme together with its cofactor(s) is called a holoenzyme (this is the active
form). Most cofactors are not covalently attached to an enzyme, but are very tightly bound. However,
organic prosthetic groups can be covalently bound (e.g., thiamine pyrophosphate in the enzyme
pyruvate dehydrogenase). Therefore,
Apoenzyme + Coenzyme/cofactor → Holoenzyme
Antienzyme: Antienzymes are the substances produced as a result of repeated injection of certain
enzymes in the serum, which can prevent the normal action of enzymes injected e.g. anti-pepsin, anti-
tripsin, anti-renin etc.

17.Differentiate between enzyme and coenzyme.
Followings are the differences between enzyme and coenzyme:
Enzyme Coenzyme
Enzyme is a organic catalyst. Coenzyme is accessory substances.
Enzyme is protein in nature. Coenzyme is non-protein in nature.
Enzyme is non-dialyzable. Coenzyme is dialyzable.
Enzyme is heat and 𝑝𝐻 liable. Coenzyme is heat and 𝑝𝐻 stable.
Enzyme can act itself. Coenzyme is required by an enzyme
for its catalytic reaction.
18.What is the difference between prosthetic group and coenzyme?
Prosthetic group is tightly bound to the enzyme, whereas coenzymes exist in the solution in free
state and contact the enzyme only at the time of reaction.
19.What do you mean by activators? What are metals that act as coenzyme?
Activators: In some instances, the enzyme action is activated by some metallic ions, known as
activators. Metals that act as coenzymes are:
i. 𝑍𝑛++
ion for carbonic anhydrase,
ii. 𝐶𝑢++
ion for tyrosine,
iii. 𝐹𝑒++
ion for catalase peroxidase,
iv. 𝑀𝑔++
ion for pyrophosphatase and
v. 𝑀𝑛++
ion for aminopeptidase.
20.Distinguish between hormone & enzyme.
Differences between hormone and enzyme are given below:
Hormone Enzyme
Hormone is a chemical substance that is secreted
into the body fluid by one cell or a group of cells
and that exert physiological control effect on
other cells of the body.
It is an organic catalyst produces by the living cell.
Hormone is steroid, peptide, protein or amino
acid in nature.
Enzyme is protein in nature.
Hormone secretion depends upon releasing or
inhibitory hormone from the hypothalamus.
Enzyme activity depends upon substrate
concentration, 𝑝𝐻, temperature.
Controlled by (secretion or production) feedback
mechanism.
Controlled by hormone, physical or chemical
effects.
It does not act as a catalyst. Enzyme acts as a catalyst.
Transported in the blood throughout the body
and acts far from the site of origin.
Acts at the site where it is produced but some
(e.g. digestive enzymes) function outside their cell
of origin.
Hormone exhibits a high degree of specificity of
action, only target cells will respond to their
hormones.
Enzyme exhibits a relative or absolute specificity
of action.

21.Discuss the uses of enzymes as therapeutic agents.
Enzymes as therapeutic agents:
 Streptokinase prepared from streptococcus is useful for clearing the blood clots. Streptokinase
activates plasma plasminogen to plasmin which, in turn, attacks fibrin to convert into soluble products.
Plasminogen
↓ 𝑆𝑡𝑟𝑒𝑝𝑡𝑜𝑘𝑖𝑛𝑎𝑠𝑒
Plasmin
↓
Fibrin (clot) → Solubleproducts
 The enzyme aparaginase 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.
 Enzyme is used in heart stimulating injection such as inj. digitoxin, inj. digotoxic etc. It is made
from cardiac glycoside which is extracted from plant digitalis.
Following a list of therapeutic application of enzymes is given:
Enzyme Therapeutic application
Streptokinase/urokinase To remove blood clot.
Asparaginase In cancer therapy.
Papain Anti-inflammatory.
𝛼1-antitrypsin To treat emphysema (breathing difficulty due to distension of lungs).
22.Discuss diagnostic importance of enzyme.
What are the clinical diagnoses of enzymes?
Estimation of enzyme activities in biological fluids (particularly plasma/serum) is of great clinical
importance. Enzymes in the circulation are divided into two groups – plasma functional and plasma
non-functional.
Estimation of the activities of plasma non-functional enzymes is very important for the diagnosis
and prognosis of several diseases.
The normal serum level of an enzyme indicates the balance between its synthesis and release in
the routine cell turnover. The raised enzyme levels could be due to cellular damage, increased rate of
cell turnover, proliferation of cells, increased synthesis of enzymes etc. Serum enzymes are
conveniently used as markers to detect the cellular damage which ultimately helps in the diagnosis of
diseases.
A summary of the important enzymes useful for the diagnosis of specific diseases is given in
following table:
Serum enzyme (elevated) Disease (most important)
Amylase Acute pancreatitis
Serum glutamate pyruvate transaminase (SGPT) Liver diseases (hepatitis)
Serum glutamate oxaloacetate transaminase (SGOT) Heart attacks (myocardial infarction)
Alkaline phosphatase Rickets, obstructive jaundice
Lactate dehydrogenase (LDH) Heart attacks, liver diseases
Creatine phosphokinase (CPK) Myocardial infarction (early marker)
Alsolase Muscular dystrophy
5’-neucleotidase Hepatitis
𝛾-glutamyl transpeptidase (GGT) Alcoholism

Detailed information on the diagnostic enzymes including reference values is provided in following
table:
Enzymes Reference value Disease(s) in which increased
Digestive enzymes:
Amylase
Lipase
2.5 − 5.5 𝜇𝐾𝑎𝑡
0.2 − 1.5 𝐼𝑈/𝐿
Acute pancreatitis, mumps (acute parotitis),
obstruction in pancreatic duct, severe
diabetic ketoacidosis.
Acute pancreatitis, moderate elevation in
carcinoma of pancreas.
Transaminases:
Alanine transminase (ALT)
or SGPT
Asparatate transminase
(AST) or SGOT
3 − 40 𝐼𝑈/𝐿
4 − 45 𝐼𝑈/𝐿
Acute hepatitis (viral or toxic), jaundice,
cirrhosis of liver.
Myocardial infarction, liver diseases, liver
cancer, cirrhosis of liver.
Phosphatases:
Alkaline phosphatase (ALP)
Acid phosphatase (ACP)
In adults 25 − 90 𝐼𝑈/𝐿
2.5 − 12 𝐼𝑈/𝐿
Bone diseases (related to higher osteoblastic
activity) – rickets, Pagets’ disease,
hyperparathyroidism, carcinoma of bone.
Prostatic carcinoma i.e. cancer of prostate
gland, Pagets’ disease.
Enzymes of carbohydrate
metabolism:
Aldolase
Isocitrate dehydrogenase
(ICD)
Lactate dehydrogenase
(LDH)
2 − 6 𝐼𝑈/𝐿
1 − 4 𝐼𝑈/𝐿
50 − 200 𝐼𝑈/𝐿
Mascular dystrophy, liver diseases,
myocardial infarction, myasthenia gravis,
leukemias.
Liver diseases (inflammatory toxic or
malignant).
Myocardial infarction, acute infective
hepatitis, macular dystrophy, leukemia,
pernicious anemia.
Miscellaneous enzymes:
Creatine kinase (CK) or
creatine phosphokinase
(CPK)
Choline esterase (ChE I)
5’-neucleotidase or
nucleotide phosphatase
(NTP)
𝛾-glutamyl transpeptidase
(GGT)
Ceruloplasmin
(ferrooxidase)
10 − 50 𝐼𝑈/𝐿
2 − 10 𝐼𝑈/𝐿
2 − 15 𝐼𝑈/𝐿
5 − 40 𝐼𝑈/𝐿
20 − 50 𝑚𝑔/𝑑𝐿
Myocardial infarction, muscular dystrophy,
hypothyroidism, alcoholism.
Nephritic syndrome, myocardial infarction.
Hepatitis, obstructive jaundice, tumors.
Alcoholism, infective hepatitis, obstructive
jaundice.
Bacterial infections, collagen diseases,
cirrhosis, pregnancy.
In following table decreased plasma enzymes in certain disorders are given:
Enzyme Reference values Disease(s) in which decreased
Amylase 80 − 180 𝑆𝑜𝑚𝑜𝑔𝑦𝑖
𝑢𝑛𝑖𝑡𝑠/𝑑𝐿
Liver diseases.
Pseudocholine esterase
(ChE II)
10 − 20 𝐼𝑈/𝑑𝐿 Viral hepatitis, malnutrition, liver cancer,
cirrhosis of liver.
Ceruloplasmin 20 − 50 𝑚𝑔/𝑑𝐿 Wilson’s disease.

Glucose 6-phosphate
dehydrogenase (G6PD) in
RBC
120 − 260 𝐼𝑈/1012
RBC Congenital deficiency with hemolytic anemia
23.What is pancreatin?
Pancreatin: Pancreatin is a mixture of several digestive enzymes produced by exocrine cells of the
pancreas. It is composed of amylase, lipase and protease. This mixture is used to treat conditions in
which pancreatic secretion are deficient. It is given in mouth in the treatment of pancreatic deficiency
associated with condition such as cystic fibrosis and pancreatitis. It is denatured by acid and high
doses, in entericoated dosage form, must be given to overcome the 𝑝𝐻 of the stomach.
Pancreatin hydrolyses fat, glycerol and fatty acids; break down proteins into peptides, proteases
and derived substances and converts starch into dextrin and sugar. It is available in the market in the
form of powder, capsules, containing powder on entericoated granules.
24.Describe your understanding about enzymatic hydrolysis of proteins.
The dietary protein first reaches in the stomach where it is hydrolyzed by pepsin. Pepsin hydrolyses
the dietary protein into a mixture of polypeptide.
Dietary protein
𝑝𝑒𝑝𝑠𝑖𝑛
→ Polypeptide
The polypeptides formed thus in the stomach are digested in the intestine by tripsin, chymotrypsin
and carboxy peptidases secreted in pancreatic juice and amino peptidases present in the intestinal
mucosa. Tripsin hydrolyses peptide linkage containing arginine or lysine and chymotrypsin hydrolyses
peptide linkages containing tyrosine or phenylalanine.
Polypeptides
𝑡𝑟𝑦𝑝𝑠𝑖𝑛 𝑎𝑛𝑑 𝑐ℎ𝑦𝑚𝑜𝑡𝑟𝑦𝑝𝑠𝑖𝑛
→ Peptides + Amino acids
Carboxy peptidase-𝐴 hydrolyses the end group of peptides containing aromatic or aliphatic amino
acid and releases free amino acids. Carboxy peptidase-𝐵 hydrolyses peptides containing arginine and
lysine residues. The intestinal mucosa also contains tripeptidase, dipeptidase etc. which hydrolyze tri-
peptide and di-peptide respectively.
Peptides
𝑐𝑎𝑟𝑏𝑜𝑥𝑦 𝑝𝑒𝑝𝑡𝑖𝑑𝑎𝑠𝑒
→ Amino acids
Peptides
𝑎𝑚𝑖𝑛𝑜𝑝𝑒𝑝𝑡𝑖𝑑𝑎𝑠𝑒
→ Amino acids
Di-peptides
𝑑𝑖𝑝𝑒𝑝𝑡𝑖𝑑𝑎𝑠𝑒
→ Amino acids
Tri-peptides
𝑡𝑟𝑖𝑝𝑒𝑝𝑡𝑖𝑑𝑎𝑠𝑒
→ Amino acids
Thus the final products of digestion of protein are amino acids which are absorbed in the body.
25.Discuss the role of enzyme in the process of fermentation to produce alcohol.
Enzymes play a great important role to produce alcohol in the process of fermentation. In this
process the enzyme diastase is used to hydrolyze starch to maltose. To the solution of maltose yeast is
added. The enzymes maltase and zymase are present in the yeast. The enzyme maltase converts
maltese into glucose and zymase converts glucose into alcohol.
2(𝐶6 𝐻10 𝑂5) 𝑛 + 𝑛𝐻2 𝑂
𝑑𝑖𝑎𝑠𝑡𝑎𝑔𝑒
→ 𝑛(𝐶12 𝐻22 𝑂11)
Maltose
𝑚𝑎𝑙𝑡𝑎𝑠𝑒
→ Glucose + Fructose
Glucose
𝑧𝑦𝑚𝑎𝑠𝑒
→ Alcohol + 𝐶𝑂2

26.Mention some industrial applications of enzymes.
Enzymes are used in the chemical industry and other industrial applications when extremely
specific catalysts are required. Followings are the industrial applications of enzymes:
Application Enzymes used Uses
Food processing
Amylases from fungi and
plants
Production of sugars from starch, such as in
making high-fructose corn syrup. In baking,
catalyze breakdown of starch in the flour to sugar.
Yeast fermentation of sugar produces the carbon
dioxide that raises the dough.
Proteases
Biscuit manufacturers use them to lower the
protein level of flour.
Baby foods Trypsin To predigest baby foods.
Brewing industry
Enzymes from barley are
released during the
mashing stage of beer
production
They degrade starch and proteins to produce
simple sugar, amino acids and peptides that are
used by yeast for fermentation.
Industrially produced
barley enzymes
Widely used in the brewing process to substitute
for the natural enzymes found in barley.
Amylase, glucanases,
proteases
Split polysaccharides and proteins in the malt.
Betaglucanases and
arabinoxylanases
Improve the wort and beer filtration
characteristics.
Amyloglucosidase and
pullulanases
Low-calorie beer and adjustment of
fermentability.
Proteases
Remove cloudiness produced during storage of
beers.
Acetolactatedecarboxylase
(ALDC)
Increases fermentation efficiency by reducing
diacetyl formation.
Fruit juices Cellulases, pectinases Clarify fruit juices.
Jeans fading Per oxidase In pumic wash.
Dairy industry
Rennin, derived from the
stomachs of young
ruminant animals (like
calves and lambs)
Manufacture of cheese, used to hydrolyze protein.
Microbially produced
enzyme
Now finding increasing use in the dairy industry.
Lipases
Is implemented during the production of
Roquefort cheese to enhance the ripening of the
blue-mould cheese.
Lactases Break down lactose to glucose and galactose.
Meat tenderizers Papain To soften meat for cooking.
Starch industry
Amylases,
amyloglucosideases and
glucoamylases
Converts starch into glucose and various syrups.
Glucose isomerase
Converts glucose into fructose in production of
high fructose syrups from starchy materials. These
syrups have enhanced sweetening properties and
lower calorific values than sucrose for the same

level of sweetness.
Paper industry
Amylases, Xylanases,
Cellulases and ligninases
Degrade starch to lower viscosity, aiding sizing and
coating paper. Xylanases reduce bleach required
for decolorising; cellulases smooth fibers, enhance
water drainage, and promote ink removal; lipases
reduce pitch and lignin-degrading enzymes
remove lignin to soften paper.
Biofuel industry
Cellulases
Used to break down cellulose into sugars that can
be fermented (see cellulosic ethanol).
Ligninases Use of lignin waste.
Biological
detergent
Primarily proteases,
produced in an
extracellular form from
bacteria
Used for presoak conditions and direct liquid
applications helping with removal of protein stains
from clothes.
Amylases
Detergents for machine dish washing to remove
resistant starch residues.
Lipases
Used to assist in the removal of fatty and oily
stains.
Cellulases Used in biological fabric conditioners.
Contact lens
cleaners
Proteases
To remove proteins on contact lens to prevent
infections.
Rubber industry Catalase
To generate oxygen from peroxide to convert latex
into foam rubber.
Photographic
industry
Protease (ficin)
Dissolve gelatin off scrap film, allowing recovery of
its silver content.
Molecular biology
Restriction enzymes, DNA
ligase and polymerases
Used to manipulate DNA in genetic engineering,
important in pharmacology, agriculture and
medicine. Essential for restriction digestion and
the polymerase chain reaction. Molecular biology
is also important in forensic science.
Soap and detergent
industry
Lipase
Used to assist in the removal of fatty and oily
stains.
Amylase
Detergent for machine dish washing to remove
resistance starch residues.
Cellulase Used in biological fabric conditions.
27.What are the biological roles of enzymes?
The enzymes have many applications in our daily life. The manifold applications are described
below:
i. Enzymes serve a wide variety of functions inside living organisms. They are indispensable for
signal transduction and cell regulation, often via kinases and phosphatases. They also generate
movement, with myosin hydrolysing ATP to generate muscle contraction and also moving cargo
around the cell as part of the cytoskeleton. Other ATPases in the cell membrane are ion pumps
involved in active transport.
ii. Enzymes are also involved in more exotic functions, such as luciferase generating light in
fireflies. Viruses can also contain enzymes for infecting cells, such as the HIV integrase and reverse
transcriptase or for viral release from cells, like the influenza virus neuraminidase.
iii. An important function of enzymes is in the digestive systems of animals. Enzymes such as
amylases and proteases break down large molecules (starch or proteins, respectively) into smaller

ones, so they can be absorbed by the intestines. Starch molecules, for example, are too large to be
absorbed from the intestine, but enzymes hydrolyse the starch chains into smaller molecules such as
maltose and eventually glucose, which can then be absorbed. Different enzymes digest different food
substances. In ruminants which have a herbivorous diets, microorganisms in the gut produce another
enzyme, cellulase to break down the cellulose cell walls of plant fiber.
iv. Several enzymes can work together in a specific order, creating metabolic pathways. In a
metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the
catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one
enzyme can catalyze the same reaction in parallel, this can allow more complex regulation: with for
example a low constant activity being provided by one enzyme but an inducible high activity from a
second enzyme.
v. Enzymes determine what steps occur in these pathways. Without enzymes, metabolism
would neither progress through the same steps, nor be fast enough to serve the needs of the cell.
Indeed, a metabolic pathway such as glycolysis could not exist independently of enzymes. Glucose, for
example, can react directly with ATP to become phosphorylated at one or more of its carbons. In the
absence of enzymes, this occurs so slowly as to be insignificant. However, if hexokinase is added, these
slow reactions continue to take place except that phosphorylation at carbon 6 occurs so rapidly that if
the mixture is tested a short time later, glucose-6-phosphate is found to be the only significant
product. Consequently, the network of metabolic pathways within each cell depends on the set of
functional enzymes that are present.
vi. Enzymes are used in the treatment of blood clot. Example: urokinase is used in the treatment
of blood clot in brain.
vii. Enzymes help to make building tissues.
viii. Enzymes take part in energy producing reactions.
ix. Cheese making: Enzymes play an important role in cheese making. They help in coagulating
the casein in milk.
x. The enzymes are being used for processing fruits juice such as apple juice, grape juice etc.
xi. Enzymes help in leather processing by de-haring hide.
xii. Certain enzymes are used in chemical analysis. Example: urease is used for determination of
urea in blood.
xiii. Enzymes play an important role in fermentation process to produce alcohol.
28.What are the physiological roles of enzyme?
Followings are the physiological roles of enzymes:
i. Enzymes play an important in role in protein hydrolysis. The enzymes involved in protein
hydrolysis are trypsin, peptidese, amino peptidese etc.
ii. Oxidoreductase enzymes are involved biological oxidation and reduction. For example
dehydrogenase enzyme catalyses the removal of hydrogen and vice versa.
|
𝑂𝐻
+ 𝑁𝐴𝐷+
𝒍𝒂𝒄𝒕𝒂𝒕𝒆 𝒅𝒆𝒉𝒚𝒅𝒓𝒐𝒈𝒆𝒏𝒂𝒔𝒆
↔
𝐶𝐻3 − 𝐶 − 𝐶𝑂𝑂−
||
𝑂
+ 𝑁𝐴𝐷𝐻 + 𝐻+
Lactate ion Pyruvate ion
iii. Hexocinase enzyme catalyses the inter conversion of glucose and ATP with glucose-6-
phosphate and ADP.
𝐴𝑇𝑃 + glucose
↔ 𝐴𝐷𝑃 + glucose-6-phosphate
iv. Enolase enzyme catalyses a step of glycolysis, reversible dehydration of 2-phosphate
glycerate to phosphenol phyruvate.
2-phosphoglycerate
𝒆𝒏𝒐𝒍𝒂𝒔𝒆
↔ phosphophenol pyruvate

v. Virtually every biochemical reaction is catalyzed by enzyme. So enzyme regulates life.
29.What are the environmental benefits of enzymes?
Environmental benefits of enzymes are given below:
a. Replacing acids, alkalis or oxidizing agents in fabric desizing e.g., amylase.
b. Use of enzymes in the tanneries to reduce the use of sulphides.
c. Enzymes replace pumic stones for ‘stone washing’ of jeans. This reduces pumic waste.
d. Enzymes used in animal feeds allow more complete digestion of feed leading to less animal
waste per pound gained.
e. Replacing acids in the starch processing industry.
starch
𝒂𝒎𝒚𝒍𝒂𝒔𝒆
↔ glucose + arabinose
f. Using of enzymes in industry reduce industrial waste.
30.Discuss the role of enzyme as analytical reagent.
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.
31.Discuss the thermodynamics of enzymatic reaction.
The enzyme catalyzed reactions may be broadly grouped into three types based on
thermodynamic (energy) considerations.
 Isothermic reactions: The energy exchange between reactants and products is negligible e.g.
glycogen phosphorylase.
Glycogen + 𝑃𝑖 → Glucose 1 − phosphate
 Exothermic (exergonic) reactions: Energy is liberated in these reactions e.g. urease.
Urea → 𝑁𝐻3 + 𝐶𝑂2 + energy
 Endothermic (endergonic) reactions: Energy is consumed in these reactions e.g. glucokinase.
Glucose + 𝐴𝑇𝑃 → Glucose 6 − phosphate + 𝐴𝐷𝑃
32.Short note: Coenzyme.
Definition: Many enzymes catalyze reactions of their substrates only in the presence of specific
heat-stable, low molecular weight organic molecules called coenzyme. We get a factor or group
without amino acid when we hydrolyze protein. This factor is called coenzyme. The coenzyme e.g.
𝐾+
, 𝐹𝑒3+
etc. activates apoenzyme.
Coenzymes are heat-stable, dialyzable, non-protein organic molecules and the prosthetic groups of
enzymes.
Classification: Coenzyme is classified as following:
1. Based on chemical characteristics: Coenzymes are classified into 3 groups e.g.
a. Containing an aromatic hetero ring:
i. 𝐴𝑇𝑃 and its relatives,
ii. 𝑁𝐴𝐷, 𝑁𝐴𝐷𝑃,
iii. 𝐹𝑀𝑁, 𝑇𝑃𝑃, 𝐵6 − 𝑃𝑂4.

b. Containing a non-aromatic hetero ring:
i. Biotin,
ii. Lipoic acid.
c. Containing no hetero ring:
i. Sugar phosphate,
ii. Coenzyme 𝑄.
2. Based on functional characteristics: Coenzymes are classified into 2 groups e.g.
a. Hydrogen transferring coenzymes:
i. 𝑁𝐴𝐷, 𝑁𝐴𝐷𝑃,
ii. 𝐹𝐴𝐷, 𝐹𝑀𝑁,
iii. Coenzyme,
iv. Lipoid acid.
b. Group transferring (other than hydrogen) coenzymes:
i. 𝐴𝑇𝑃 and its relatives,
ii. 𝑇𝑃𝑃,
iii. Coenzyme 𝐴,
iv. Sugar phosphate,
v. 𝐵6 − 𝑃𝑂4,
vi. Biotin and
vii. Lipoic acid.
3. Based on nutritional characteristics:
a. Containing vitamin 𝐵-complexes:
i. Coenzyme 𝐴,
ii. 𝑇𝑃𝑃,
iii. 𝑁𝐴𝐷, 𝑁𝐴𝐷𝑃,
iv. 𝐵6 − 𝑃𝑂4,
v. Folic acid coenzyme,
vi. 𝐵12 coenzyme,
vii. Biotin,
viii. FMN.
Properties (characteristics):
 Coenzymes are heat stable and non-protein organic molecules.
 Coenzymes are dialyzable (non-colloid).
 Coenzymes are derivatives of vitamin 𝐵-complex.
 Coenzymes participate in:
o Hydride (𝐻) and electron transfer reactions e.g. 𝑁𝐴𝐷, 𝑁𝐴𝐷𝑃, 𝐹𝑀𝑁, 𝐹𝐴𝐷.
o Group transfer reactions e.g. coenzyme 𝐴, 𝑇𝑃𝑃, 𝐵6 − 𝑃𝑂4.
Functions:
 Coenzymes usually accept atoms or groups from a substance and transfer them to other
molecules.
 Coenzymes are less specific than enzymes and the same Coenzymes can act as such in a
number of different reactions.
 Coenzymes are also attached to the protein at a different but adjacent site, so as to be in a
position to accept atoms or groups that are removed from the substrate.
 Some Coenzymes performs specific function e.g.
 NAD and NADP acts as hydrogen acceptor in dehydrogenation reactions.
 Coenzyme A carry acyl groups and they are used in the oxidative decarboxylation of
pyruvic acid and synthesis of fatty acids and acetylation.

 TPP carry active aldehyde group.
 𝐵6 − 𝑃𝑂4 involves in transamination reactions.
33.Coenzymes are considered as second substrate (or co-substrate) – explain.
Coenzymes are considered as second substrate for the following reasons:
 Coenzyme involved reactions are of very great physiological significance. Example: in the
anaerobic glycolysis, during muscular exercise, the reaction converting pyruvic acid into lactic acid
causes reoxidation of 𝑁𝐴𝐷𝐻 to 𝑁𝐴𝐷. This 𝑁𝐴𝐷 then permits synthesis of 𝐴𝑇𝑃.
 The chemical lchanges in the coenzyme exactly counterbalance those taking place in the
substrate. Example: in oxidoreduction (dehydrogenase) reactions, one molecule of substrate is
oxidized (dehydrogenated) and one molecule of coenzyme is reduced (hydrogenated).
Viva Special
 Enzyme is protein in nature (exception – 𝑅𝑁𝐴 acting as ribozyme).
 A for digit Enzyme Commission (EC) number is assigned to each enzyme representing the class
(first digit), sub-class (second digit), sub-sub-class (third digit) and the individual enzyme (fourth digit).
 Enzyme is 10,000 − 1,00,000 times faster or effective than catalyst.
 Enzyme is used in biological detergent (Surf Excel).
 Enzyme is used as bighting agent in detergents, cosmetics etc. (protein absorbs 280 𝑛𝑚 UV
light.
 Enzyme is used to test diabetic. Usual test cannot determine 𝛼 − 𝐷 − glucose. All 𝛼 − 𝐷 −
glucose is first converted into 𝛽 − 𝐷 − glucose with the help of mutarotase. Then 𝛽 − 𝐷 − glucose is
estimated by glucose oxidase.
 Action of enzyme is very specific. There is only one chance of doing wrong in 1,00,00,000
times.
 Urea is used to transform curly protein to linear protein structure. Urea makes bond with
hydrogen, therefore, the protein is straightened down.
 When it is vitamin is called coenzyme and when it is organic compound or metal is called
cofactor or prosthetic group.
 Apoenzyme + Coenzyme = Holoenzyme
Protein part Non-protein part
 As enzymes are made of protein they are non-dialyzable.
 If the reaction is enzyme catalyzed or not can be differentiates by two tests: (i) heat sensitive
test and (ii) acid test.
 The name ligase comes from Greek ligate – to bind.
 𝐾 𝑚 = Michaelis-Menten constant or Brig’s and Haldane’s constant. It is defined as that
substrate concentration which produces half of the maximum velocity. It is defined in terms of
substrate concentration. Low 𝐾 𝑚 is preferred because at low concentration we can achieve the
maximum velocity.
 Two categories of enzymes requires metals for their activity such as (i) metal-activated
enzymes (not tightly held with metal) e.g. ATPase ( 𝑀𝑔++
, 𝐶𝑎++
), enolase ( 𝑀𝑔++
) and (ii)
metalloenzymes (tightly held with metal) e.g. phenol oxidase (𝐶𝑢), pyruvate oxidase (𝑀𝑛), xanthine
oxidase (𝑀𝑜), cytochrome oxidase (𝐹𝑒, 𝐶𝑢).
 Suicide inhibition is a specialized form of irreversible inhibition where the original inhibitor is
converted to a more potent form by the same enzyme that ought to be inhibited.
 Specificity is a characteristic property of the active site.

 The specificity of the enzyme is mostly dependent on the apoenzyme and not on the
coenzyme.
 Mechanism of enzyme action:
o Lock and key model or Fischer’s template theory,
o Induced fit theory or Koshland’s theory,
o Substrate strain theory and
o Michaelis-Menten theory.
 Mechanism of enzyme catalysis:
o Acid-base catalysis,
o Substrate strain,
o Covalent catalysis and
o Entropy effects.
 Regulation of enzyme activity in the living system:
o Allosteric regulation,
o Activation of latent enzymes,
o Compartmentation of metabolic pathways,
o Control of enzyme synthesis,
o Enzyme degradation and
o Isoenzymes.
 The existence of life is unimaginable without the presence of enzymes – the biocatalysts.
 Majority of the Coenzymes (𝑇𝑃𝑃, 𝑁𝐴𝐷+
, 𝐹𝐴𝐷, 𝐶𝑜𝐴) are derived from 𝐵-complex vitamins in
which form the later exert their biochemical functions.
 Competitive inhibitors of certain enzymes are of great biological significance. Allopurinol,
employed in the treatment of gout, inhibits xanthine oxidase to reduce the formation of uric acid. The
other include aminopterin used in the treatment of cancers, sulfanilamide as antibactericidal agent
and dicumarol as an anticoagulant.
 Enzyme inhibitors exert their effect by acting on the apoenzyme, coenzyme, prosthetic group
or activators present in the enzyme system or by interfering with the binding of substrate to the
enzyme.
 Feedback (or end product) inhibition is a specialized form of allosteric inhibition that controls
several metabolic pathways e.g. 𝐶𝑇𝑃 inhibits aspartate transcarbamolyase; cholesterol inhibits 𝐻𝑀𝐺
coenzyme 𝐴 reductase. The end product inhibition is utmost important to cellular economy since a
compound is synthesized only when required.
 Allosteric site is the site other than active site which regulates the activity of the enzyme. It
binds with other than the substrate. The compound that binds with it is known as allosteric effector. A
modifier may binds with it.
 The nerve gas (diisopropyl fluorophosphates), first developed by Germans during Second
World War, inhibits acetylcholine esterase, the enzyme essential for nerve conduction and paralyses
the vital body functions. Many organophosphorus insecticides (e.g. melathion) also block the activity
of acetylcholine esterase.
 Penicillin antibiotics irreversibly inhibit serine containing enzymes of bacterial cell wall
synthesis.
 Certain 𝑅𝑁𝐴 molecules (ribozymes) function as non-protein enzymes. It is beloved that
ribozymes were functioning as biocatalysts before the occurrence of protein enzymes during
evolution.
 Isoenzymes are the multiple forms of an enzyme catalyzing the same reaction which however,
differ in their physical and chemical properties. 𝐿𝐷𝐻 has five isoenzymes while 𝐶𝑃𝐾 has three.
 Turnover number: The combined processes of enzyme synthesis and degradation constitute
enzyme turnover.
 Process of enzyme isolation:

o Dialysis,
o Absorption,
o Differential heat or 𝑝𝐻 denaturation,
o Differential centrifugation,
o Gel filtration,
o Electrophoresis and
o Precipitation with varying salt concentration.
 Units of enzyme activity:
o 𝐾𝑎𝑡𝑎𝑙: One 𝑘𝑎𝑡 denotes the conversion of one mole substrate per second (𝑚𝑜𝑙/𝑠𝑐𝑒).
Also 𝑚𝑘𝑎𝑡, 𝜇𝑘𝑎𝑡 and so on.
o International unit (𝐼𝑈): One 𝐼𝑈 denotes the amount of enzyme activity that catalyses
the conversion of one micromol (𝜇𝑚𝑜𝑙) of substrate per minute.
o 1 𝐼𝑈 = 60 𝜇𝑘𝑎𝑡
o 1 𝑛𝑘𝑎𝑡 = 1.67 𝐼𝑈
 Coenzyme 𝐴: It is composed of 𝐴𝑇𝑃, pantothenic acid and 𝛽-mercaptoethylamine. So it is the
coenzyme form of pantothenic acid.
 Coenzyme 𝑄: Coenzyme 𝑄 is a derivative of benzoquinone in which one of the substitute is
polyisoprenoid chain. It is also known as ubiquinone.
 Subdivision of cofactor:
Cofactors
Organic
(coenzymes)
Tightly bound
(prosthetic
group)
Loosely
bound(second
substrate)
Inorganic (metal
ions)
Tightly bound
(metalloenzyme)
Loosely bound
(ion activators)

Notes on Enzyme

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