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Enzymology
Enzymes are biological catalysts which bring about chemical are present
in very small amounts in various cells . Almost all the functions of the
body such as digestion , breathing , synthesis and breakdown of
carbohydrates m fats and proteins are catalysed and controlled by
specific enzymes . Most chemical reactions of the living cells would have
occurred very slowly had it not catalysed by enzymes . The substance
upon which an enzymes acts is called the substrate . The enzyme will
convert the substrate into the product or products . The enzymes are
generally named by adding the suffix-ase to the name the substrate .
Thus the enzyme lactase acts on the substrate lactase and products
glucose and galactose are formed . All enzymes are proteins . Enzymes
follows the physical and chemical reaction of proteins . They are heat
labile , soluble in water , precipitated by protein precipitating reagents (
Ammonium sulphate or trichloroacertic acid ) and contain 16 % weight
nitrogen .
General Properties of Enzymes
1.All enzymes are proteins .
2. They accelerate the reaction , but
a. do not alter the reaction equilibrium
b. not consumed in overall reaction
c. required only in very small quantities.
3. They have enormous power for catalysis .
4. Enzymes are highly specific for their substrate .
5. Enzymes possess active sites at which interaction with substrate takes
place .
6. Enzymes lower activation energy .
7. They form substrate complex as intermediates during their action
8. Some enzymes are regulatory in function .
Classification of Enzymes
As per the International Union of Biochemists , enzymes are divided into
six major classes .
Oxidoreductases
One compound oxidized , another reduced , e.g. tyrosinase , urease , lactic
hydrogenase , catalase and peroxidase .
Transferase
This class of enzymes transfers group containing C , N, or S from the
substrate to another substrate substrate . They are important in is ,
biological synthesis , e.g. transaminases , hexokinases transcylases ,
transaldolases . Hydrolases
They catalyse hydrolysis of esters , ether , peptide or glycosidic bond by
addition of water molecules across the bond which is split , e.g. esterases ,
peptidases .
Lyases
They catalyse the addition or removal pf groups without hydrolysis ,
oxidation or reduction m producing double bonds at times , e.g.
decarboxylases , carboxylases , carbonic anhydrase .
Isomerases
These can produce optical , geometric or position isomers of substrates by
intermolecular rearrangement , e.g. reacemases, epimerases , isomerases .
Ligases
These enzymes also called synthetases link two substrates together
usually with the linking of pyrophosphate bound in ATP ( Ligare = to
bind ) , e.g. glutamine synthetase .
ENZYME SPECIFITY
Some enzymes are very specific and show activity with only one substrate
.Some others are much less particular . Generally three types of
enzymatic specificities are observed .
1. Sterospecificity : Some enzymes show specificities only with a specific
group of a substrate , e.g. urease catalyses the hydrolysis of urea .
O
║
Urease
H 2N –C-NH2 + 2H2O
(NH4)2 CO3
The catalysis dose not take place when structure of urea is altered , e.g. N
methhyyl urea thiourea are not hydrolysed by urrase .
O
S
║
║
H2N –C-NH-CH3
H2N-C-NH2
N-Methyl Urea
Thiourea
D – amino acid – oxidase acts only on the D- form of amino acid and not
on L- form .
2. Substrate specificity : Some enzymes catalyes similar type of reaction
but differ in their action due to substrate specificity , e.g. (a) Pepsin
hydrolyses peptide bonds involving aromatic amino acids like phenyl
alanine and tyrosine . (b) Trypsin hydrolyses peptide bonds involving
carboxyl group of basic amino acids like arginine or lysine .
3. Reaction specificity : A substrate can undergo many reactions but in
reaction specificity , one enzyme can catalyse only one of the various
reaction . For example , oxalic acid can undergo different reactions , but
each of these reactions is catalysed by separate enzyme .
Mechanism of Enzyme Acids
According to Michaelis and Menten , the enzyme molecule (E) first
combines with a substrate molecule (S) to form an enzyme substrate (ES)
complex which further dissociates to form product (P) and enzyme (E)
back :
E + S ES E + products
Enzymes once dissociated from the complex if free to combine with
another molecule of the substrate . The site at with a substrate can meet
with the enzyme molecule is extremely specific and is called active site or
catalytic site . Normally the molecule size and shape of the substrate
molecule is extremely small compared to the enzymes molecules . The
active site is made up of several amino acid residues that come together as
a result of the folding of secondary and tertiary structures of the enzymes
. So the active site possesses a complex three dimensional form and shape
, provides a predominantly non-polar cleft or crevice to accept and bind
the substrate .
Factors Affecting Enzymes Activity
Substrate Concentration
At a low substrate concentration the initial velocity of an enzymes
catalysed reaction is proportional to the substrate concentration.
However , as the substrate concentration is increased , the initial velocity
increases less as it is no longer proportional to the substrate
concentration . With a further increase in the substrate concentration
and the velocity assumes a constant rate as a result of enzymes being
saturated with its substrate . It was Michaelis and Menten who suggested
an explanation for these findings by postulating that at low substrate
concentrations , the enzyme is not saturated with the substrate and the
reaction is not proceeding at maximum velocity whereas when the
enzymes is saturated with substrate , maximum velocity is observed . The
enzymes combines with the substrate to form an enzyme- substrate
complex and the rate of decomposition of the substrate is proportional to
the enzyme – substrate complex. The substrate proportional to the
enzyme – substrate complex . The velocity of the reaction at this high
substrate concentration is termed as maximum velocity (Vmax) . The
velocity is called the Michael's constant (Km) . Km indicates the affinity
of the substrate towards the enzymes and is inversely proportional to the
affinity .
1
Km = α
Affinity
Higher the affinity the smaller will be the Km and lower the affinity , the
higher will be the Km ( Fig .7.1) . The Michaelis – Menten equation is
given by the expression
Vmax [S]
V=
Km + [S]
Where V = Initial velocity
Vm= Maximum velocity
Km = Michaela's constant
[S] = Substrate concentration
When the initial velocity is exactly half the maximum velocity ,
Vmax [S]
Km + [S]
2
Km + [S] = 2[S]
1
Vmax
=
Km = [S]
VMAX
V
E
L
O
C
I
T
Y
1/ 2 vmax
Substrate concentration
Fig . 7.1 : Enzyme activity and substrate concentration
Therefore , Km is equal to substrate concentration at which the velocity is
half the maximum .
Effect of Enzyme concentration
The rate of an enzyme catalysed reactioly is direct proportional to the
concentration of the enzyme. The greater the concentration of enzyme ,
the faster will be reaction taking places( Fig 7.2).
Effect of pH
The enzyme activity is maximum at a particular pH which is called the
optimum pH . This id due to the change in the net
Reaction
rate
Substrate concentration
Fig . 7.2: Enzyme activity and enzyme concentration
Charge on enzymes resulting from change in pH. Excessive changes in pH
brought on by addition of strong acids or bases may completely and
inactivate enzymes ( Fig. 7.3) .
Optimum
pH
Reaction
rate
0
7
14
pH
Fig .7.3. : Enzyme activity and pH
Effect of Temperature
The velocity of enzyme reaction increase when temperature of the
medium is increased ; reaches a maximum and then falls . The
temperature at which maximum amount of substrate is converted to the
product per unit time is called the optimum temperature ( Fig . 7.4).
Reaction
rate
Denaturation
Optimum
Temperature
0
20
40
60
Fig.7.4: Enzyme activity and temperature
As temperature is increased, more molecules get activation energy, or
molecules are at increased rate of motion, and so their collision
probabilities along with the reaction rate is increased.
Above this temperature the reaction rate decreases as enzymes being
protein in nature are denatured by heat and becomes inactive.
Effect of Time
The time required for completion of an enzyme reaction increases with
decrease in temperature from its optimum. Under the opti-mum
conditions of pH and temperature, time required for enzyme reaction is
less.
Enzyme Inhibition
Enzyme are proteins and they can be inactivated by the agents that
denature them. The chemical substances which inactivate the enzymes
are called as inhibitors and the process is called enzyme inhibition. Most
of the substances commonly referred to as poisons are harmful in that
they inhibit one or more essential enzymes.
The inhibitors may be classified in two broad groups. First,compounds
or ions which are specific in their effect, inhibiting only one enzyme or
several closely related enzymes. And second, substances which are
nonspecific, inhibiting many enzymes.
Specific Inhibition
The inhibitor molecule is a structural analog of the normal substrate of
the enzyme, i.e. it is chemically similar to the substrate. The inhibitor is
capable of combining with the active site by virtue of its similar structure.
As long as the active site is bound to the inhibitor, the enzyme is not a
catalyst. Since both are competing for combination with same active site,
the term competitive inhibition is often used.
An important example is provided by the sulfa drugs. These are
structural analongs of paraaminobenzoic acid, a compound required by
many bacteria for certain metabolic processes but which is not used by
higher organisms such as man. The sulfa drugs inhibit growth of the
bacteria in man by competing with the paraaminobenzoic acid for the
active site of some bacterial enzyme.
NH2
NH2
O=S=O
C=O
OH
p-aminobenzoic Acid
NH2
Sulfanilimide
Non-Specific Inhibition
Every protein molecule has a number of reactive groups present on side
chains of the constituent amino acids, groups such as –CO2H, -SH, -NH2,
etc. Any substance capable of combining with a common group of this
type is a potential inhibitor of all.
Heavy metal ions are non-specific inhibitors. They can bind to a number
of protein groups in particular –SH and –CO2H at the active site
inhibiting the normal catalytic. In sufficient concentration, heavy metal
ions will inhibit most enzymes and are therefore poisonous to all living
things. However, some of these heavy metal ions Cu++,Zn++, Co++ are
absolute requirements in low concentrations for cells as cofactors for a
variety of enzymes. Thus classification of a metal ion as poison depends
on its concentration. Mostly non-specific inhibition is non-compe-titive in
nature.
CO-ENZYMES
Many enzymes require the presence of small non-protein organic
molecules for their efficient performances. Only when both enzy-me
and co-enzyme are present catalysis will occur. Co-enzymes are low
molecular weight, non-protein organic
compounds that are heat
resistant, function as cosubstances. Usually, it binds loosely and can be
easily separated from its enzyme by dialysis, but when it binds tightly , it
is considered as a prosthetic group of the enzyme . Co – factor differs
from a co – enzyme only because it usually is a metallic ion ( Fe , Mg , Zn
, Cu ) rather than an organic molecule.
The term apo-enzyme refers to the protein part of the enzyme.
The apo-enzyme with its prosthetic group (or co-enzyme) consti-tute a
complete enzyme or holoenzyme.
Conjugated protein enzyme
protein + prosthetic group
Or
Holoenzyme
Apo-enzyme + co+enzyme
=Protein part + non-protein part (prosthetic group)
Classification of Co-enzyme
Co-enzymes for group transfer
Co-enzymes for transfer of H
1.ATP and its relatives
1. NAD+ , NADP+
2. Sugar phosphates
2. FMN, FAD
3. CoA
3. Lipoic acid
4. Thiamine pyrophosphate
4. Co-enzyme Q
5. B6 phosphate
6. Folate co-enzyme
7. Biotin
8. Cobamide (B12) Co-enzyme
9. Lipoic acid
Water Soluble Vitamins as Co- Enzymes
Vitamins of B complex and vitamin C ( Ascorbic acid )act as co-enzymes
as shown in Table 7.1
Vitamins
Thiamine (B1)
Co- enzymes
Thiamine pyrophosphate TPP
Riboflavin (B2)
Flavin mononucleotide ( FMN)
Flavin adenine dinucleotide
(FAD)
Pyridoxal phosphate
Pyridoxine(B3)
Niacin (B3)
Din
Pantothenic acid
Biotin
E
Types of reaction
Decarboxylation of α
- keto acid certain
reaction of ketosugars
Several kind of
Oxidation – reduction
reactions.
Several kinds of
Reactions involving
Amino acids , e.g.
decarboxylation
transamination.
Nicotinamide adenine
Numerous oxidation
dinucleotide ( NAD+)
- reduction reaction
Nicotinamide adenine dinucleotide
phosphate (NADP+)
co-enzyme 'A'
Enzyme bound biotin
Many reaction of fatty
Acids , particularly
those involving transfer
of acetyl groups .
Certain carbon dioxide
Folic acid
Tetrahydrofolic acid
Cyanoco-balamine Several " Cobamide "
co-enzymes
fixation reactions.
Various reactions
involving single carbon
compounds
Carbon chain
Isomerizations , certain
methyl group transfers.
Diagnostic Value of Plasma Enzymes
When a tissue is injured some cells of that tissue are destroyed and their
contents including enzymes are released into the blood stream . The
increase of enzymes in blood stream will indicate the disease . ( See Table
7.2) .
Table 7.2: Increase of Different Enzymes in Diseases
Enzymes
Amylase
Acid Phosphatase(Optimum
pH = 5
Alkaline phosphatase ( Optimum
pH=10
Aspartate transaminase AST
( previously GOT )
Alanine transaminase ALT
( previously GPT)
Lactase dehydrogenase LDH
Creatine kinase (CK)
Γ Glutamyl transferase (γGT)
Urinary Elevation
Increased in
Acute pancreatitis
Prostatic carcinoma.
Liver disease , rickets .
Myocardial infarction.
Liver disease especially with
liver cell damage
Myocardial infarction but also
increased in liver disease and
some blood disease .
Myocardial infarction and
skeletal muscle disease
( muscular dystrophy ,
dermatomytomyositis ) .
Diagnosis of liver diseases ,
particularly biliary obstruction
and alcoholism .
N acetyl glucosaminidase in the urine can be used to indicate renal
transplant rejection .