Beruflich Dokumente
Kultur Dokumente
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.
Nature: Enzymes are protein in nature. So they can be inactivated or destroyed by excessive heat
and are precipitated by salts of heavy metals.
Enzymes are made of protein. As proteins cannot be dialyzed, so enzymes are not dialyzable.
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).
DHN CHANDAN – Department of ACCT, RU (Mobile – 88-01713413431) 1
16/07/08 Enzyme Pharmaceutical
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 − 𝒍𝒂𝒄𝒕𝒂𝒕𝒆 𝒅𝒆𝒉𝒚𝒅𝒓𝒐𝒈𝒆𝒏𝒂𝒔𝒆
𝐶𝐻3 − 𝐶 − 𝐶𝑂𝑂−
| + 𝑁𝐴𝐷+ → || + 𝑁𝐴𝐷𝐻 + 𝐻 +
𝑂𝐻 𝑂
Lactate ion Pyruvate ion
ii. Alcohol dehydrogenase oxidizes alcohol to aldehyde or ketone.
𝒂𝒍𝒄𝒐𝒉𝒐𝒍 𝒅𝒆𝒉𝒚𝒅𝒓𝒐𝒈𝒆𝒏𝒂𝒔𝒆
Alcohol + 𝑁𝐴𝐷+ → 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− 𝐶𝑂𝑂−
| |
+
𝐶=𝑂 𝐶𝑂𝑂− 𝐻3 𝑁 − 𝐶 − 𝐻 𝐶𝑂𝑂 −
| | | |
𝒂𝒎𝒊𝒏𝒐𝒕𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒂𝒔𝒆
𝐶𝐻2 + 𝐻3 𝑁 − 𝐶 − 𝐻 →
+
𝐶𝐻2 + 𝐶 = 𝑂
| | | |
𝐶𝐻2 𝑅 𝐶𝐻2 𝑅
| |
−
𝐶𝑂𝑂 𝐶𝑂𝑂−
α-ketoglutaric acid glutamate α-keto acid
ii. Serine hydroxyl-methyl transferase catalyzes the transfer of carbon.
− −
𝐶𝐻2 − 𝐶𝐻 − 𝐶𝑂𝑂 𝐶𝐻2 − 𝐶𝑂𝑂 𝑇𝐻𝐹
𝒔𝒆𝒓𝒊𝒏𝒆 𝒉𝒚𝒅𝒓𝒐𝒙𝒚−𝒎𝒆𝒕𝒉𝒚𝒍 𝒕𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒂𝒔𝒆
| | + 𝑇𝐻𝐹 → | + \ /
+ + 𝐶𝐻2
𝑂𝐻 𝑁𝐻3 𝑁𝐻3
Serine Glysine
iii. Choline acyl transferase catalyzes the transfer of acyl-containing group.
𝒄𝒉𝒐𝒍𝒊𝒏𝒆 𝒂𝒄𝒆𝒕𝒚𝒍 𝒕𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒂𝒔𝒆
acetyl-𝐶𝑜𝐴 + choline → 𝐶𝑜𝐴 + 𝑂-acetyl choline
iv. Hexokinase catalyzes transfer of phosphorous containing group.
𝒉𝒆𝒙𝒐𝒌𝒊𝒏𝒂𝒔𝒆
𝐴𝑇𝑃 + 𝐷-hexose → 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.
ATP ADP + Pi
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
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.
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.
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.
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:
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.
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.
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.
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.
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 𝑂
| ||
𝒐𝒙𝒊𝒅𝒂𝒔𝒆
𝑅 − 𝐶 − 𝐶𝑂𝑂𝐻 → 𝑅 − 𝐶 − 𝐶𝑂𝑂𝐻
|
𝑁𝐻2
𝐻
|
𝒅𝒆𝒄𝒂𝒓𝒃𝒐𝒙𝒚𝒍𝒂𝒔𝒆𝒆
𝑅 − 𝐶 − 𝐶𝑂𝑂𝐻 → 𝑅 − 𝐶𝐻2 − 𝑁𝐻2 + 𝐶𝑂2
|
𝑁𝐻2
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.
𝒖𝒓𝒆𝒂𝒔𝒆
𝐻2 𝑁 − 𝐶𝑂 − 𝑁𝐻2 + 𝐻2 𝑂 → 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.
14 DHN CHANDAN – Department of ACCT, RU (Mobile – 88-01713413431)
16/07/08 Enzyme Pharmaceutical
glucose 𝒉𝒆𝒙𝒐𝒌𝒊𝒏𝒂𝒔𝒆 glucose-6-phosphate
} + 𝐴𝑇𝑃 → } + 𝐴𝐷𝑃
fructose fructose-6-phosphate
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,
DHN CHANDAN – Department of ACCT, RU (Mobile – 88-01713413431) 15
16/07/08 Enzyme Pharmaceutical
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).
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.
+𝑆
𝐸 ↔ 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.
Substrate: Substrate means the main chemical compound which undergoes alteration in a
chemical reaction upon which the enzyme exerts its influence.
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 + , 𝐹𝑒 3+ 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.
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.
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.
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.
The dietary protein first reaches in the stomach where it is hydrolyzed by pepsin. Pepsin hydrolyses
the dietary protein into a mixture of polypeptide.
𝑝𝑒𝑝𝑠𝑖𝑛
Dietary protein → 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.
𝑡𝑟𝑦𝑝𝑠𝑖𝑛 𝑎𝑛𝑑 𝑐ℎ𝑦𝑚𝑜𝑡𝑟𝑦𝑝𝑠𝑖𝑛
Polypeptides → 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.
𝑐𝑎𝑟𝑏𝑜𝑥𝑦 𝑝𝑒𝑝𝑡𝑖𝑑𝑎𝑠𝑒
Peptides → Amino acids
𝑎𝑚𝑖𝑛𝑜𝑝𝑒𝑝𝑡𝑖𝑑𝑎𝑠𝑒
Peptides → Amino acids
𝑑𝑖𝑝𝑒𝑝𝑡𝑖𝑑𝑎𝑠𝑒
Di-peptides → Amino acids
𝑡𝑟𝑖𝑝𝑒𝑝𝑡𝑖𝑑𝑎𝑠𝑒
Tri-peptides → 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.
𝑑𝑖𝑎𝑠𝑡𝑎𝑔𝑒
2(𝐶6 𝐻10 𝑂5 )𝑛 + 𝑛𝐻2 𝑂 → 𝑛(𝐶12 𝐻22 𝑂11 )
𝑚𝑎𝑙𝑡𝑎𝑠𝑒
Maltose → Glucose + Fructose
𝑧𝑦𝑚𝑎𝑠𝑒
Glucose → Alcohol + 𝐶𝑂2
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
Production of sugars from starch, such as in
making high-fructose corn syrup. In baking,
Amylases from fungi and
catalyze breakdown of starch in the flour to sugar.
plants
Food processing Yeast fermentation of sugar produces the carbon
dioxide that raises the dough.
Biscuit manufacturers use them to lower the
Proteases
protein level of flour.
Baby foods Trypsin To predigest baby foods.
Enzymes from barley are They degrade starch and proteins to produce
released during the simple sugar, amino acids and peptides that are
mashing stage of beer used by yeast for fermentation.
production
Industrially produced Widely used in the brewing process to substitute
barley enzymes for the natural enzymes found in barley.
Amylase, glucanases, Split polysaccharides and proteins in the malt.
proteases
Brewing industry
Betaglucanases and Improve the wort and beer filtration
arabinoxylanases characteristics.
Amyloglucosidase and Low-calorie beer and adjustment of
pullulanases fermentability.
Remove cloudiness produced during storage of
Proteases
beers.
Acetolactatedecarboxylase Increases fermentation efficiency by reducing
(ALDC) diacetyl formation.
Fruit juices Cellulases, pectinases Clarify fruit juices.
Jeans fading Per oxidase In pumic wash.
Rennin, derived from the Manufacture of cheese, used to hydrolyze protein.
stomachs of young
ruminant animals (like
calves and lambs)
Microbially produced Now finding increasing use in the dairy industry.
Dairy industry
enzyme
Is implemented during the production of
Lipases 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.
Amylases, Converts starch into glucose and various syrups.
amyloglucosideases and
glucoamylases
Starch industry Converts glucose into fructose in production of
high fructose syrups from starchy materials. These
Glucose isomerase
syrups have enhanced sweetening properties and
lower calorific values than sucrose for the same
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
24 DHN CHANDAN – Department of ACCT, RU (Mobile – 88-01713413431)
16/07/08 Enzyme Pharmaceutical
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.
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.
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
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 + , 𝐹𝑒 3+ etc. activates apoenzyme.
Coenzymes are heat-stable, dialyzable, non-protein organic molecules and the prosthetic groups of
enzymes.
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.
DHN CHANDAN – Department of ACCT, RU (Mobile – 88-01713413431) 27
16/07/08 Enzyme Pharmaceutical
TPP carry active aldehyde group.
𝐵6 − 𝑃𝑂4 involves in transamination reactions.
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.
𝐾𝑚 = 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 𝐾𝑚 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.
28 DHN CHANDAN – Department of ACCT, RU (Mobile – 88-01713413431)
16/07/08 Enzyme Pharmaceutical
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:
DHN CHANDAN – Department of ACCT, RU (Mobile – 88-01713413431) 29
16/07/08 Enzyme Pharmaceutical
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
Loosely bound
(ion activators)