• +919785333312/14
  • info@letstalkacademy.com

LET'S TALK LIFE-SCIENCE BIOCHEMISTRY

Suraj Prakash Sharma | Ekta Chotia

KINETIC ANALYSIS
46

8.6.3.     Uncompetitive Inhibition

There is no competition between inhibitor and the substrate as sites of attachments of the substrate and inhibitor are different. Inhibitor has no structural similarity to substrate therefore cant bind to free enzyme. Inhibitors binds with enzyme substrate complex that expose inhibitor binding site. Binding of inhibitor can cause distortion of the active site or allosteric site that inactivates the catalysis.

Effect on affinity :

High affinity of inhibitor means low dissociation of enzyme substrate complex to enzymes substrate. In this, inhibitor binds to other then active site on enzyme substrate complex. That means inhibitor show affinity for ES complex rather then enzyme. Thus in the presence of inhibitor the affinity of enzyme toward substrate increases. This decrease the Km. Therefore

Effect on Vmax

Vmax is calculated at infinite substrate concentration. At infinite substrate concentration all enzymes are in the form of enzymes substrate complex. The inhibitor show affinity for enzymes substrate complex. Thus inhibitor binds to enzymes substrate complex and prevent the catalysis of enzyme substrate complex into enzyme and product. That's why Vmax decreases and new Vmax is given by

Inhibitor concentration increases a value increases and Vmax decreases

On putting the values of new Km and  new Vmax in lineweaver burk plot, The equation is as follows :-

Uncompetitive inhibitor causes different intercepts on both Y and X-axis but same slope.

8.6.4.     Mixed (Non-Competitive) Inhibition

This inhibitor is not similar to substrate structurally but can binds to free enzyme and the enzyme substate complex both.

When inhibitor binds to the enzyme away from the active sites. It induces the conformational changes and reduces its catalytic activity. Thus, enzyme inhibitor [EI] and enzyme substrate inhibitor [ESI] complexes become non productive. The substrate concentration does not reverse the reaction. Hence, inhibition leads to unaltered Km but reduced Vmax.

Lineweaver Burk plot is used to determine Km and Vmax in enzyme kinetics. The Y-intercept of such a graph is equivalent to the inverse of Vmax, X intercept of the graph represents competitive inhibitors hence the same Y-intercepts (as Vmax is unaffected by competitive inhibitors) but there are different slops.

Non competitive inhibitor produces plot with same X-intercept as Km is unaffected but different slopes with Y-intercepts.

8.7.         Kinetics of Multisubstrate Reaction

In the enzyme kinetics, simple reactions involve one substrate binding to an enzyme and undergoing catalytic reactions. This condition is not common. A majority of biochemical reactions catalyzed by two or more substrates taking part in the reactions. For example, an enzyme E, catalyzed the reaction involving two substrates A and B and yield the product P and Q.

This type of reaction is called as Bi-Substrate reaction . These reaction can proceed in two ways:

8.7.1.     Sequential

Both the substrates A and B, bind to the enzyme E, and then reactions proceeds to yield products P and Q

This type of reaction is called as sequential or simple-displacement reactions which are further divided into following groups.

Ordered substrate binding or ordered sequential mechanism – In this type, one substrate must bind before a second substrate.

This reaction indicates the sequential binding of substrates as well as sequential release of product. This type of mechanism is observed in the reactions catalyzed by lactate dehydrogenes involving NAD+ and lactate.

8.7.2.     Random substrate binding- In this type either A or B may bind to the enzyme first, followed by the other substrate and the release of the product.

This type of mechanism is observed in reactions catalyzed by transferases enzyme as hexokinase catalyzed phosphorylation of glucose by ATP.

8.7.3.     Theorell-Chance Sequential mechanism

It is a type of ordered sequential bisubstrate reaction in which the ternary complex does not accumulate.

8.7.4.     Ping pong mechanism

The other possibility in bi-substrate reaction is that one substrate, A, binds to the enzyme and on reacting with it a product, P, is released and enzyme turns into a modified form, E′. The second substrate, B, comes in and binds with modified enzyme to yield second product, Q and regenerate the enzyme, E.

The reactions following the above mechanism are called Ping-Pong or double-displacement reactions. This type of mechanism is observed in reactions catalyzed by aminotransferases.

These enzymes catalyze the transfer of an amino group from an amino acid to an α-keto acid.The products formed are a new amino acid corresponding to keto acid and a new keto acid corresponding to carbon skeleton of amino acid such as:-

Another example of ping pong reaction is phosphoglycerate mutase. The enzyme get phosphate from one substrate and after phosphorylation of enzyme, the phosphate is transferred to second substrate.

Page no. 46