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BIOMOLECULES
Active Site: The active site (or active centre) of an enzyme is defined as small region at which the
substrate binds and participates in the catalysis.
- The existance of active site is due to the tertiary structure of protein resulting in three dimensional
native conformation. Loss of native enzyme structure (say by denaturation) will result in derangement
of active site.
- Active sites are regarded as clefts/crevices or pockets occupying a small region in a big enzyme
molecule.
- Active site is not rigid in structure and shape. It is rather flexible to promote specific substrate binding.
- Active site possesses a substrate binding site (Buttress site) and a catalytic site.
- The coenzyme or cofactors on which some enzyme depend are present as a part of the catalytic
site.
Enzyme Kinetics
Km Value: Michaelis-Menten Constant [Brig’s and Haldane’s constant]
Km : amount of substrate required to attain half maximum velocity.
1
Km
Affinity/activity of enzyme
Michaelis Menten Equation
V [S]
V= max
Km + [S]
V = Measured Velocity
Vmax = Maximum Velocity
S = Substrate concentration
Km = Michael is Menten constant
Enzyme Inhibition
Enzymes are sensitive to inhibitors.
Inhibitors may be organic or inorganic in nature.
Enzyme inhibitions are reversible inhibition and irreversible inhibition.
Enzyme inhibitions are
(i) Competitive inhibition (ii) Non Competitive inhibition
(iii) Uncompetitive inhibition
(i) Competitive inhibition : Also called substrate analogue inhibition.
- In competitive inhibition, the inhibitor bears a close structural similarity with the substrate and competes
the substrate for active site and forms enzyme-inhibitor complex.
- The relative concentration of substrate and inhibitor and their respective affinity with the enzyme
determines the degree of competitive inhibition.
- The inhibition could be overcome by high substrate concentration.
- In competitive inhibition, Km value increases (increases by Ki) and Vmax remains unchanged.
Examples
Enzyme Substrate Competitive inhibitor
Succinate dehydrogenase Succinate Malonate
Aconitase cis-Aconitate tans-Aconitate
Lactate dehydrogenase Lactate Oxamate
- Competitive inhibitors are often used in the control of Bacterial Pathogens.
[For eg. Sulpha drugs [Sulphanilamide] inhibit the synthesis of Folic acid in bacteria by competing
with para-aminobenzoic acid (PABA) for active site of enzyme.
Non Competitive Inhibition
In this case inhibitor do not complete with substrate for active site. Binding of inhibitor to enzyme
(any where except active site) disturbs conformation / structure of enzyme that results disturbance of
catalytic site (no effect on Buttress site) i.e. will not effect binding affinity (no effect on Km) but will
decrease product formation (Vmax decreases).
[Inhibitors can bind to free enzyme and ES complex]
Example
Heavy metal ions (Ag+, Pb+2, Mg+2 etc.) can non competitively inhibit enzymes.
Uncompetitive Inhibition : In this case inhibitor neither binds with free enzyme nor inhibit the formation
of ES complex. On the contrary, the inhibitor binds with only the ES complex and thereby enhances the
substrate affinity of the enzyme to lower Km.
Inhibitor always binds to ES complex to retard product formation i.e. Vmax decreases.
Revessible Inhibition : Inhibitor binds non covalently with enzyme and inhibition can be reversed.
Irreversible Inhibition : Inhibitors binds covalently with the enzyme and inactivate them.
Example:
• Iodoacetate.
• Diisopropyl flurophosphate (DFP) is nerve gas (Developed by Germans during Second World
War).
• Many organophosphorus insecticides like melathion are toxic to animals as they block the
activity of acetylcholine esterase (essential for nerve conduction) resulting in paralysis of vital
body functions.
• Penicillin antibiotic-Irreversibly inhibit enzyme involved in Bacterial Cell Wall Synthesis.
Allosteric enzyme
These enzyme are usually oligomeric
These enzymes have a specific site for modulator called allosteric site. Modulator which binds at
allosteric site can effect binding of substrate to active site in either way.
Allosteric site is different from active site.
These enzymes usually shows product feed back inhibition. Some shows substrate feedback
inhibition.
Enzyme kinetic curve is sigmoid (S-shaped).
Exist in 2 forms R(Relaxed) form & T form (tensed form)
Glucose Hexokinase Glucose-6-Phosphate
ADP
ATP
Fructose 6-Phosphate Phosphofructokinase Fructose 1,6-Diphosphate
ATP ADP
Excess of ATP inhibit phosphofructokinase (this is substrate feed back)
