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Factors influencing enzyme activity
The activity of enzymes is markedly affected by several factors. These factors are
3. substrate concentration
4. metal ions (activators)
6. enzyme concentration etc.
All the enzymes have a particular pH at which their activity is maximal; above or below this pH the activity is low. The pH at which the enzyme shows maximum activity is known as optimum pH. Some of the enzymes and their optimum pH are
(a) Pepsin - 2.0
(b) Urease - 7.0
(c) Salivary amylase - 6.8
(d) Alkaline phosphatase - 9.9
Only in this optimum pH, ionisation of active amino acids in enzymes and substrate are favoured for ES complex formation.
The pH activity relationship is shown in the Fig. 3.1.
Rise in temperature causes increase in the rate of enzyme catalysed reactions up to a certain temperature i.e about 45°C. Above which the activity declines due to denaturation of enzymes (due to their protein nature). As the enzyme is denatured and inactivated, the reaction which it catalyses slows down and ultimately stops. So the temperature at which the enzyme shows maximum activity is known as optimum temperature. The optimum temperature of most of the enzymes is found to be 37°C. The relationship of enzyme activity to temperature is shown below in Fig. 3.2:
With a fixed amount of enzyme, the reaction rate is proportional to the concentration of substrate. But this is true upto a certain concentration after which the increase in concentration of substrate does not further increase the velocity of the reaction.
Since the number of active sites on an enzyme molecule are limited, a stage will come when all of them have filled with the substrate molecules. This is known as saturation of enzyme. Now, since none of the active sites of the enzyme is free, further addition of the substrate molecule will not increase the product formation (Fig.3.3).
It was Michaelis and Menten in 1913, who proposed a successful explanation for the effect of substrate concentrtaion on the enzyme activity. According to them the enzyme ‘E’, and the substrate ‘S’ combine rapidly to form a complex, the enzyme substrate complex ‘ES’. The complex then breaks down relatively, slowly to form the product of the reaction. The enzyme regenerated can involve in another round of catalysis.
E + S < - - > ES
ES - - > E + P
Divalent ions, like Mg2+, Cu2+, Mn2+, Zn2+ and monovalent ions such as Na+ and K+ are required for the activity of many enzymes. For example, amylases need Cl- ions, Zn2+ ions are required for carbonic anhydrase action, Fe2+ and Cu2+ ions are required for enzymes involved in redox reactions. Several peptidases are activated by Mn2+, Zn2+ or Co2+. Enzymes requiring metal ions or enzymes which contain metal ions in their structure are called as metallo enzymes.
The velocity of an enzymatic reaction is directly proportional to the concentration of enzyme. In case the enzyme concentration is doubled then as much as twice active site become available to combine with the substrate, provided an excess of substrate is present and so the maximum velocity is also doubled. At a fixed concentration of the substrate a level is reached when all the substrate molecules are utilised and no more change in velocity of the reaction takes place (Fig. 3.4).
Chemical substances which reduce the activity of enzymes are called as inhibitors. They may be small inorganic ions such as cyanide which inhibits the enzyme cytochrome oxidase or much more complex molecules such as diisopropyl phospho fluoridate which inhibit acetyl choline esterase.
This phenomenon in which the enzyme activity is decreased by the presence of inhibitors is known as enzyme inhibition.
Enzyme inhibition may be of different types such as
(c) non-competitive and
(d) allosteric inhibition.
This type of inhibition occurs when the structure of inhibitor resembles that of the substrate. The inhibitor competes with the proper substrate for binding at the active site of the enzyme. In this type of inhibition, both ES complex and EI complex (enzyme - inhibitor complex) are formed during the reaction. The relative amounts of the two complexes depend partly upon the affinity of the enzyme towards the substrate and inhibitor and partly upon the relative concentration of substrate and the inhibitor. Thus if the inhibitor is present in sufficiently high concentration, it can displace the substrate entirely and thus blocks the reaction completely (Fig.3.5).
Succinate dehydrogenase catalyses the conversion of succinic acid to fumaric acid
This reaction is completely inhibited by malonic acid which has structural resemblence with succinic acid.
- a competitive inhibitor of succinate dehydrogenase
This type of inhibition can be reduced by increasing the concentration of the substrate and for this reason competitive inhibition is called as reversible inhibition. Many competitive inhibitors are used as drugs to block particular metabolic reactions.
In this type of inhibition, the inhibitor combines with enzyme - substrate complex to give an inactive enzyme - substrate - inhibitor complex which cannot undergo further reaction to yield the product (Fig. 3.6).
In this type, the degree of inhibition may increase when the substrate concentration is increased. This inhibition cannot be reversed by increasing the concentration of substrate.
In this type of inhibition, the inhibitor can combine with either the free enzyme or the enzyme substrate complex, interfering with the action of both. Non competitive inhibitor bind to the site on the enzyme other than the active site, often to deform the enzyme, so that it does not form the ES complex at its normal rate and once formed, the ES complex does not decomposes at the normal rate to yield products. These effects are not completely reversed by increasing the substrate concentration (Fig. 3.7).
a. Effect of iodoacetamide on - SH group containing enzymes
b. Effect of diisopropyl phosphofluoridate on acetyl choline esterase.
These two inhibitors completety inactivate the respective enzymes.
This inhibition can be partially reversible.
This type of inhibition is otherwise known as end product inhibition. The inhibitor binds with the modulator binding site (or) allosteric site of the enzyme. The inhibitor present at the allosteric site may affect the conformation at the active site with the result it becomes difficult for the enzyme to take up the substrate molecule, and in the extreme case, the enzyme completely fails to take up the substrate molecule (Fig. 3.8).
This type of inhibition is seen in multistep reactions in which each step is catalysed by different enzymes as shown below.
where A is the starting substrate B,C,D,F are intermediates, a,b,c,d are enzymes and P the product. When the product concentration (P) increases, it binds with the enzyme ‘a’ which is the first enzyme in the reaction sequence. This enzyme which can be inhibited by the end product is known as allosteric enzyme.
when isoleucine production increases, as a regulatory mechanism, it binds with threonine deaminase in the allosteric site and inhibit further binding of the substrate with the enzyme and ultimately production of isoleucine is stopped. This inhibition is otherwise known as feed back inhibition.
Many metabolic reactions in our body are regulated by means of allosteric enzymes.
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