Tuesday, 15 December 2020

Factors Affecting Enzyme Activity

  Enzymes cause blochemical transformations within the cell. by following rules of chemical kinetics. A number of factors are known to interfere with activity of enzymes. They are as under: 

1. Concentration of substrate
2. Concentration of Enzyme
3. Temperature of Incubation 
4. pH
5. Time of incubation 
6. State of oxidation
7. Concentration of end product

8. Presence of effectors.

These factors influence activity of enzyme by causing
a). Change in velocity of enzyme reaction.
b). Interfering with the product produced.
c). Change in maximum velocity attained by enzyme.

1). Effect of substrate concentration

  The substance on which enzyme acts, is called substrate. It is normally found that Increase in substrate concentration causes Increase in velocity of enzyme reaction. Generally, In lower range of substrate concentration, the increase in substrate concentration results in proportional increase in velocity (v) of enzyme.
  However, at higher range of substrate concentration, this proportionality is not maintained. At a particular concentration of substrate, enzyme attains maximum reaction velocity (Vmax). Further increase in substrate concentration may not cause increase in velocity. Thus it gives parabolic curve.

The behavior of enzyme in this way is due to following reasons:

a). Initlally, Increase in substrate concentration facilitates rapid formation of enzymes-substrate (ES) complex. This is due to increased chances for the random collision between enzyme and substrate molecules.  
   This, therefore, reduces the time for conversion of substrate into product. This decrease in time for ES complex formation will continue up to a certain level as the substrate concentration Increases.

b). At high substrate concentrations, however, a minimum time may be required for ES complex formation, and definite time is required for formation of product. Therefore, beyond a particular substrate concentration. further Increase in velocity is not possible.

  The rate of in velocity of enzyme reaction with the increase in substrate concentration depends on affinity of enzyme for the substrate. Higher is the affinity, more rapid is the formation of ES complex and hence rapid is the Increase in V of enzyme. Therefore, enzymes with higher affinity for substrate can show increase in V even at low substrate concentration.
This relationship between affinity of enzyme for substrate and V is expressed as Km and Vmax values. Km is the Michaelis-Menten constant. Vmax is the maximum velocity of the enzyme.

2). Effect of Enzyme Concentration

  Increase in concentration of enzyme causes increase in velocity of enzyme reaction. As the enzyme concentration
Increases, velocity of enzyme reaction also increases up to a certain concentration of enzyme.


At a particular enzyme concentration, It achieves maximum velocity. Such behavior of enzyme is due to following reasons:
a). Increase in enzyme concentration causes increased chances for random collision with substrate. This Increases the rate of ES complex formation. Hence it causes increase in velocity of enzyme reaction.
b). Higher concentration of enzyme allows more ES complex formation and so substrate is rapidly utilized.
c). However, at particular concentration of enzyme, all substrate will be utilized within the period of incubation and so no further substrate is left out to give more products. At this potnt enzyme will show maximum velocity.

3). Effect of Temperature

Like all chemical reactions, enzyme catalyzed reactions are also influenced by temperature. With every Increase in temperature by 10°C, there is a two fold Increase in velocity of enzyme reaction. This continues up to a certain temperature. Further increase beyond this temperature will cause decrease in enzyme reaction velocity.
  The temperature at which enryme has maximum velocity is called optimum temperature. The temperature during which velocity increases is called temperature of activation and temperature during which velocity decrease is called temperature of inactivation.

This Influence of temperature on enzyme reaction is due to following reasons:

a). Initlally increase in temperature causes increase In kinetic energy of enzyme and substrate molecules. So collision between enzyme and substrate becomes rapid. This favors rapid formation of ES complex.

b). Temperature also increases the reaction energy and so conversion; E. S. ➞  E. P. ➞ E + P become rapid. Hence, velocity of enzyme reaction increases.

c). Enzyme proteins are thermo labile. Therefore, beyond certain temperature, the enzyme proteins coagulate and denature. This accounts for Inactivation of enzyme reaction.

4). Effect of pH

At a particular value of pH. the enzyme shows maximum activity. This is known as optimum pH for enzyme. The change in pH on elther side of this causes decrease in velocity of enzyme reaction. The range of pH, between which, enzyme shows activity is called pH range for enzyme.

  This behavior of enzyme is due to amphoteric nature of enzyme protein, and the change in their conformation due to change in pH.

5). Effect of time

  It is found that the amount of product produced by enzyme Increases with the increase in time of incubation. This increase continues up to certain period of incubation. However, after a definite time of incubation, no further Increase in the amount of product is observed. This time is called optimum time of Incubation.

  This is due to complete utilization of substrate by enzyme or due to establishment of state of equilibrium as enzyme reactions are reversible, except for few Irreversible enzyme reactions.

6). Effect of end product Concentration

   The increase in the concentration of end product of enzyme reaction causes inhibition in enzyme reaction. As a result, velocity as well as amount of product produced decreases. This is due to carly establishment of state of equilibrium as well as role of end product as feedback Inhibitor, which inhibits activity of enzyme.

7).  Effect of state of oxidation

The activity of enzyme is also affected by the state axidation of enzyme. Some enzymes are active under oxidized conditions, while some remain active under reduced conditions. This depends upon nature of
1. Reaction catalyzed by enzyme. 2. Active site.
e.g. Many enzyme possess ûáSH group at active site. They will be active only in reduced conditions. Reduced conditions protect -SH group form oxidation. Oxidizing conditions modify úaSH group to S-S bond and make enzyme inactive.

8). Effectors

Effectors are the small chemical molecules which modify the activity of enzymes. Based on their influence on enzyme activity, they are of two types:
a. Activators
b. Inhibltors


  Activators are the molecules which activate the enzyme activity. They act in various ways.
a). By protecting the active site of
b). By participating in formation
      of active site.
c). By facilitating the ES complex
      formation and activity of
d). By modifying protein structure
      to make it active enzyme.

a) By protecting active site
  Many effectors protect active site from environmental Influence and thus work as activators. e.g. Glutathione; -SH group of active site of enzyme ana thus work as activator of such enzymes.

b). By participating in formation of active site
Most activators fall in this group. They include vitamins and coenzymes such as NAD, NADP, FAD, FMN, Biotin, TPP, Lipolc acid, Pyridoxal PO4, ete.
In metalloenzymes, metal lons, help in formation of active site.

c). By helping formation of ES complex
May metal ions, at concentrations, help in formation of ES complex also facilitate the activity of enzyme. This include
  Kinases     : Mg++ and Ca++
  Enolase     : Mg++
  Peptidase : Co++, Mn++, Zn++

d). By modifying protein structure   
  This kind of effect is typically observed as zymogen activation. Zymogen activation is the type of enzyme activation where one enzyme can activate the other enzyme. The enzyme, which acts as activator is called zymogen.
   Here, the activity of zymogen modifies the structure of other enzyme and exposes functional group to render It active. e.g.
• Trypsinogen is converted into Trypsin by enzyme Enterokinase . • Plasminogen is converted into  Plasmin by enzyme Thrombin.

  The chemicals that inhibit the activity of enzyme are called inhibitors. They cause iInhibition by
a) Reducing the velocity of reaction
b) Decreasing the amount of product produced.

There are three major types of enzyme inhibitors and accordingly three types of Inhibitions.

Competitive Inhibitors
  These are the tnhibitors which can compete with the natural substrate of enzyme to bind at the active site. These Inhibitors are structural analogues of the substrate.
  The structural analogy allows the inhibitor to bind at active and allows formation of enzyme inhibitors (EI) complex. This decreases the probability of the formation of ES comple Therefore, the rate in increase of velocty of enzyme decreases.

The inhibition can be reverted increasing the substrate concentration. Such inhibitice therefore, Influences Km value of enzyme. Vmax remain unaffected.
e.g. Malonic acid, structural analogue of succinic acid Inhibits activity of succinic dehydrogenase.

Uncompetitive Inhibitors
   Many Inhibitors Interact directly with the functional group or active site or may Interact with other groups on the enzyme elsewhere and Interfere with the funetioning of active site in such a way that conversion of ES complex into EP comples is Interfered.
   Such Inhibitors do not interfere with ES complex formation and still effect Inhibition. Therefore, such Inhibitors are called uncompetitive inhibitors and the Inhibition is called uncompetitive inhibition. Such Inhibition allows change in Vmax without affecting Km value of enzyme. e.g. mercaptoethanol blocks enzyme by reacting with SH group at the active site.

Mixed Type Inhibition
  When the inhibitor has ability to cause inhibition, which share both competitive and non competitive inhibition characters, the inhibition is called mixed type. Here both Km and Vmax are changed due to inhibition.

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