Enzymes

• Definition-Enzymes may be defined as

biocatalyst, synthesized by living cells. They
are protein in nature( Exception-RNA acting as
ribozyme),colloidal and thermolabile in
character and specific in action.
 1 oxidoreductase
• Group of enzymes that catalyzes oxidation and
reduction reactions.
• AH2 + B --------->A +BH2
• Ex. Alcohol dehydrogenase
• Cytochrome oxidase
• D and L Amino acid oxidases
 2)transferases
• Group of enzymes that catalyzes transfer of
functional group (phosphoryl,glycosyl,methyl)
• A-X + B -----> A + B-X
• Ex.hexokinase
• transaminases
• transmethylases
• phosphorylase
 3 Hydrolases
• Group of enzymes that catalyze hydrolysis of
compounds by addition of water
• A-B + H2O ------> AH + BOH
• EX-Lipase
• Choline esterase
• Acid and Alkaline Phosphatase
• Pepsin
• urease
 4 Lyases
• Group of enzymes that catalyzes cleavage of
of bonds ( C-C,C-O,C-N) resulting in to
formation of compound having double bond
• A – X ---------> A + XY
• B Y B
• Aldolase
• Fumarase
• histidase
 5 isomerases
• Group of enzymes that catalyzes
intercovertion of stereoisomers or optical
isomers (isomerization)
• A----- A`
• Triose phosphate isomerase
• Ketoisomerase
• Retinol isomerase
• Phosphohexose isomerase
 6 Ligases
• Group of enzymes that catalyzes ligation of
two molecules (synthetic reactions)
• A + B ----> A-B
• Glutamine synthase
• Acetyl co A carboxylase
• Succinate thiokinase
 Factor affecting enzyme activity
• 1. concentration of enzyme
• 2 concentration of substrate
• 3 Effect of temperature
• 4 effect of pH
• 5 effect of product concentration
• 6 effect of activators
• 7 effect of time
8.Effect of light and radiation
 1)Effect of concentration of enzyme-as the concentration of
enzymes increased, the velocity of reaction increases
proportionately

Velocity

Concentration of enzyme
2)Increase in substrate concentration gradually increase
the velocity of enzyme reaction to certain range . Then
it becomes constant.

Effect of substrate concentration

 Enzyme kinetics and Km value
• The enzyme combines with the substrate to
form ES complex which then form product.
k1 k3
• E +S ES E +P
• k2
• K1,k2,k3 represent the velocity constant for
respective reaction
 Km (the michaelis-Menton constant) is given by the
fallowing formula
k2 +k3
• Km = ----------
k1
• The following equation is obtained after
suitable algebraic manipulation
V max[S]
• v= ----------
Km + [S]
• Where v = measured velocity
• Vmax = maximum velocity
• S = substrate concentration
• Km = Michaelis Menton Constant
• If measured velocity is half the maximum the
given equation can be written as fallow.
 Vmax = Vmax [S]
2 Km +S

Km +[S]= 2 Vmax[S]
Vmax
Km+[S] = 2[S]
Km = [S]
Where K stands for constant and m stand for Michaelis and it is defined
as substrate concentration that produced half the maximum velocity
low Km value indicated strong affinity between the enzyme and
substrate
And high Km value indicates weak affinity between enzyme and
substrate
 3)Effect of temperature –velocity of enzyme reaction
increases with increase up to maximum and then declines

Velocity

Temperature
 4) Effect of pH-increase in pH increase the enzyme activity and for most
enzymes pH is 6-8,for pepsin it is 1-2,for acid phosphatase 4-5,and for
alkaline phosphatase 10-11

Velocity

• e
 5)Effect of product concentration
• Accumulation of reaction products decreases
the enzyme activity.
• Products combines with the active site and
inhibit the enzyme activity.
E ←
• A +B = P
 6) Effect of activators
• Some of the enzyme require metallic ions for their activity
like
• Mg++,Mn++,Zn++,Ca++,Co++,Cu++,Na+,K+
• 1)Metal activated enzyme-metal is not tightly held by
enzyme(ATPase Mg++,Ca++) (enolase Mg++)
• 2)Mettalloenzymes –These enzmes hold the metal
tightly(alcohol dehydrogenase, carbonic
anhydrase,alkaline phosphastase,carboxypeptidase
contain ( zinc.) Phenol oxidase ( copper),pyruvate oxidase(
Mn)
• Xanthine oxidase (molybdenum),cytocrome oxidase (iron
and copper)
 7)Effect on time
• Under ideal and optimum conditions of pH the
time required for enzyme activity is less.
• Variation in time is observed in alteration of
pH and temperature.
 8)Effect of light and radiation

• Exposure of enzymes to ultraviolet rays beta,

gamma and x rays inactivates certain enzyme
due to formation of peroxides.
 Enzyme inhibition
• Enzyme inhibitor is defined as a substance and
binds with the enzyme and decreases the
catalytic activity of that enzyme. Inhibitor may
be organic or inorganic
 There are three broad categories of
enzyme inhibition

• 1)Reversible inhibition
• 2)Irreversible inhibition
• 3)Allosteric inhibition
 1) Reversible inhibition
• The inhibitor binds no-covalently with enzyme
and the enzyme inhibition can be reversed if
inhibitor is removed. It is of three types
• A) Competitive inhibition
• B) Non Competitive inhibition
• C) Uncompetitive inhibition
 A)Competitive inhibition
The inhibitor(i) closely resembles the substrate (s) and compete with the
sub.for active site
 The inhibition could be overcome by high
concentration of substrate. Examples completive
inhibition are
1)succinic acid-succinate dehydrogenase-Malic
acid
Malonic acid ,glutaric acid and oxalic acid are
the have similar structure with succinic acid
2)Methanol----alcohol dehydrogenase(ADH)--
formaldehyde (toxic)
Ethanol can compete with methanol for ADH
Therefore can be used in methanol poisoning
 3) Antimetabolites-are structural analogues of
substrate and thus are competitive inhibitors and used
in cancers and gout. Examples
a) Xanthine (s)-Xanthine oxidase(E)-uric acid (P)
Allopurinol (i) , so used in gout.
b) dihydrofolic acid(S)-dihydrofolate reductase(E)-
tetrahydrofolic acid (P)
Aminoterin,amethoterin,methotrexate are inhibitors of
this enzyme. Therefore used in the treatment of
leukemia and other cancers.

4) Antivitamins (sulfanilamide,dicumarol) block the

biochemical action of vitamins causing vitamin
deficiencies
• Dicumerol is a structural analog of vitamin K.
• Vitamin K is required for conversion of inactive
clotting factors to active clotting factors in
presence of carboxylase enzyme.
• Here dicumerol will appose the action of
vitamin K causing vitamin K deficiency.
B)Non competitive inhibition
 The inhibitor has no structural resemblance
with substrate. The inhibitor binds at a second
site other than the active site
E + S ------>ES-----> E+P
+ +
I I
EI +S ESI
Inhibitor binds with E as well as ES complex
• Heavy metal ions Ag+,Pb++,Hg++ can non
competitively inhibit the enzyme by binding
with cysteinyl sulfhydryl groups .General
reaction for Hg++ is
• E-SH +Hg++ ---- E—S—Hg++ +H
• Heavy metals also forms covalent bonds with
carbonyl group
 C) Uncompetitive inhibition
• The inhibitor binds to ES complex and not to
free E
• E + S----> ES -----> E+P
+
I
ESI
• Uncompetitive I has also no structural
resemblance with substrate.
 2)Irreversible inhibition
• Inhibitor binds covalently with the enzyme and
inactivate them which is irreverible
• 1)Iodoacetate is irreversible inhibitor of
enzymes like papain and glyceraldehyde 3
phosphate dehydrogenase. It combines with SH
groups of these enzymes and inactivate them
2)Diisopropyl fluorophosphates(DFP) binds with
enzymes containing serine at the active site Ex-
serine protease, acetylcholine esterase
• 3)organophosphorus insecticide like
Melathion block the activity of acetylcholine
esterase (essential for nerve conduction)
resulting in to paralysis of vital functions.
• 4)acetaldehyde ( CH3CHO)—aldehyde
dehydrogenase--->acetic acid,Disufiram
inhibits this enzyme, acetaldehyde accumulate
makes person sick
• 5) penicillin antibiotics are inhibitor of serine
containing enzymes and block the cell wall
synthesis.
• 6)Cyanide inhibits cytochrome oxidase of
ECT(binds to iron atom)
• 7)Fluoride inhibits enolase(by removing
manganese) thus inhibits glycolysis.
 Suicide inhibition
• Is a specialized form of irreversible inhibition.
• The original inhibitor is converted to more potent
form by the same enzyme.EX.
• 1)Allopurinal an inhibitor of xanthine oxidase is
converted to alloxanthine ,a more effective inhibitor
of this enzyme.
• 2)5-fluorouracil gets converted to flurodeoxyuridylate
which inhibit the enzyme thymidilate synthatase and
thus inhibit nucleotide synthesis
Some more examples of suicide inhibition

• 1)Aspirin inhibits cyclooxygenase of

prostaglandins synthesis.
• 2)Penicillin inhibits transpeptidase and blocks
bacterial wall synthesis.
• 3)Zidovudine ( AZT) inhibits enzyme reverse
transpeptidase and used in AID.
• 4)Sarin inhibits acetylcholine esterase.
 3) Allosteric inhibition
• Some enzymes possess allosteric sites (sites
other than active site)
• Enzyme activity is increased when positive
(+)allosteric effector binds to allosteric site.
• Enzymes activity is decreased when negative
(-) allosteric effector binds to allosteric site
 Enzyme inhibition by drugs
• Many of the drugs used in the treatment of
diseases acts as enzyme inhibitors
• 1) Cholesterol lowering drugs (lovastatin) inhibit
the enzyme HMG CoA reductase
• 2) Drugs like tenofovir and emtricitabine
employed to block HIV replication inhibit the
enzyme reverse transcriptase
• 3)Hypertention is treated by the drug
captopril,enalapril which inhibit angiotensin
converting enzyme.
 Enzyme specificity
• Enzymes are highly specific in their action
It is of three types
1)Stereospecificity
2)Reaction specificity
3) Substrate specificity
1)Stereospecificity or optical specificity

• Enzyme act on only one isomer

• A) L amino acid oxidase acts on L amino acid
• B) D amino acid oxidase acts on D amino acid
• C) Hexokinase acts on D hexoses
• D) Glucokinase acts on D glucose
• E) Amylase acts on alpha glycosidic linkages
• F) Cellulose acts on beta glycosidic linkages
 2)Reaction specificity
• Same substrate can undergo different types of
reaction each is catalyzed by separate enzyme.
Amino acid can undergo transamination,
oxidative deamination
,decarcoxylation,racemization
But each reaction is catalyzed by different
enzyme
 3)Substrate specificity
It is of three types

A) absolute substrate specificity

B) Relative substrate specificity
C) Broad substrate specificity
A)Absolute substrate specificity

Certain enzymes act only on one substrate

Glucokinase acts on glucose
Urease act on urea
 B)Relative substrate specificity
Some enzymes acts on structurally related
substrate.
A) Trypsin acts on peptide linkages formed by
arginine or lysine
B) Chymotrypsin acts on peptide linkages
formed phenyl alanine, tyrosine and tryptophan
C) Glycosidase act on glycosidic bond of CHO
D) Lipases acts on ester bond of lipids
 C)Broad substrate specificity
Some enzymes acts on closely related
substrates.
Hexokinase acts on
glucose,fructose,mannose,and glucosamine
But not on galactose.