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Activator: An activator is a substance, other than the catalyst or one of the substrates, that increases the rate of a catalysed reaction.
Inhibitor: An inhibitor is a substance that diminishes the rate of a chemical reaction and the process is called inhibition.
Specific Activity: The relative measure of enzyme purity in the sample, and represents the number of enzyme units per mg of protein. S.I. units - katal units per Kg of protein.
Enzyme kinetics is the study of study of the rates of chemical reactions that are catalyzed by enzymes. The rates of an enzyme catalyzed reaction generally changes with change in enzyme concentration, substrate concentration, temperature, pH, time, and presence of enzyme inhibitors. Enzymes are protein catalysts that, like all catalysts, speed up the rate of a chemical reaction without being used up in the process.
Catalyst: A catalyst is a substance, other than a reactant or a product, that enhances the rate of reaction and remains unchanged at the end of the reaction. The catalyst does not change the equilibrium constant of reaction, it only accelerates the rate of reaction in forward and reverse direction.
Assay of Enzyme Activity
For most of the enzymes, the rate of the enzyme-catalysed reaction is a function of the total enzyme concentration ([E]), concentration of substrate ([S]), and inhibitors, pH, temperature, and several other conditions. Thus measurement of initial rate of enzyme catalysed reaction, under more or less identical conditions, would be directly proportional upon the total enzyme concentration. By convention, the enzyme activity is determined in terms of rate of reaction and one international unit is defined as the amount of enzyme required to convert 1mole of substrate per minute under optimal conditions of assay. The name katal, is suggested by IUPAC - IUB, for the units of enzyme activity in S.I. system, representing transformation of 1mole of substrate per second under optimal conditions of assay. The relative measure of enzyme purity in the sample is known as specific activity, also represents the number of enzyme units per mg of proteins.
Energy of Activation
It is evident that besides the concentration of reactant, the rates of reactions are determined by rate constants. In order for a reaction to take place, colliding molecules must have sufficient energy to overcome a potential barrier known as energy of activation. The activation energy is the energy needed to form the transition state from the reactants. The transition state is unstable and will very quickly breakdown to form the products (or back to reactants), but no products can be formed from reactants unless transition state has been formed. The free energy of activation thus acts as a potential-barrier to the reaction taking place.
Many substances alter the activity of enzymes and those which tend to decrease the rate of enzyme catalysed reaction are called enzyme inhibitors. In enzyme-catalysed reactions an inhibitor frequently acts by binding to the enzyme, in which case it may be called an enzyme inhibitor
Enzyme inhibitors can be broadly divided into two groups:
Irreversible Enzyme Inhibitor
Irreversible inhibition generally results from covalent interactions and modification of enzyme. The inhibitor may act by preventing substrate - binding or it may destroy some component of catalytic site. Compounds which irreversibly denature enzyme protein or cause non-specific inactivation of the active site are not usually regarded as irreversible inhibitors. In practice, no process is irreversible but an inhibitor, which shows great affinity for the enzyme (dissociation constant in the order of 10-9 mol. l-1) is regarded as irreversible inhibitor. Example of irreversible inhibition of enzymes is inhibition of bacterial-cell-wall-synthesis enzymes by penicillin and inhibition of metal-ion requiring respiratory enzymes by cyanide.
Reversible Enzyme Inhibitor
On the basis of mechanism of inhibition, reversible inhibitors are classified into three major groups:
As the name suggests, in this case the inhibitors compete with the substrate for the binding site on the enzyme. Competitive inhibition often resembles the substrates whose reactions they inhibit, and because of this structural similarity they may compete for the same binding site on the enzyme. The enzyme bound inhibitor then either lacks the appropriate reactive groups or it is held in an unsuitable position with respect to catalytic site of enzymes or to the other potential substrates for the reaction to occur. The effect of competitive inhibitor depends upon the inhibitor concentration, the substrate concentration and the relative affinities of the substrate and the inhibitor for the enzyme. In general, at a particular inhibitor and enzyme concentration, if substrate concentration is low, the inhibitor will compete favorably with the substrate on the binding sites of the enzyme and degree of inhibition will be great. However, if at this same inhibitor enzyme concentration, the substrate concentration is high, then the inhibitor will be much less successful in competing with substrate for available binding sites and degree of inhibition will be less.
Uncompetitive bind only to enzyme substrate complex and not to the free enzyme. Substrate binding could cause a conformational change to take place in enzyme and reveal an inhibitor binding site, or the inhibitor could bind directly to the enzyme-bound substrate. In neither case does the inhibitor compete with the substrate for the same binding site, so inhibition cannot be overcome by increasing substrate concentration.
Non-competitive inhibitor can combine with enzyme molecule to produce a dead complex, regardless of whether a substrate molecule is bound or not. Hence, inhibitor must bind at different site from substrate.
Effect of pH on Enzyme Activity
The activity of majority of enzymes is often limited within a narrow pH range and the plot of activity as a function of pH yields a typical bell-shaped curve. The pH at which the enzyme exhibits maximum activity is referred to as optimum pH for that reaction. The effect of pH on enzyme activity could be ascribed to following:
The pH may alter the ionization state of reactants
H+ may be involved in the reaction and thus alter the equilibrium position of reaction.
pH might alter the ionization state of some of the side chains of amino acids essential for catalysis.
Effect of Temperature on Enzyme Activity
As the rate of chemical reaction increases with temperature, the rate of enzyme catalysed reactions also increases with increase in temperature, and the extent of enhancement depends upon the activation energy. Enzyme solutions are generally stored at low temperature (0o-4oc), activities of most of the enzymes are measured at 25o-37oC. Above certain temperature enzymes tend to loose their 3-d structure which is critical for their activity. However, there are some enzymes which cannot only withstand high temperature but are also very active at higher temperature. DNA polymerase from thermus aquaticus, used in polymerase chaise reaction is on such example.