DEPARTMENT OF BOTANY

TOPIC – Kinetics of
multisubstrates enzymatic
reactions
Submitted to – Submitted by –
Dr. Dinesh Kumar Yadav Kanchan Yadav
M.Sc 3Rd sem
Dr. Rajiv Kumar Yadav
Session 2023-24
 INTRODUCTION

 Enzyme kinetics – The discipline of understanding action mechanism of an enzyme catalyzed reaction by
determining the rate of reaction and it’s variation under experimental conditions.

 Multisubstrate enzymatic reaction – Multisubstrates reaction involved more than one substrate after
resulting in multiple products .The reaction involves a complex reaction that not only tell where the
substrate binds,but the sequence of binding as well.
For example –
 Reaction Nomenclature -

The general nomenclature has been devised to describe the number of substrates and products involved in an
enzymatic reactions using the Latin prefix uni, bi ,ter and so on to refer to one,two ,three and more chemical
entities.
 Bi Bi Reaction Mechanism
Bi bi reaction catalyzed by a single enzyme in which two
substrates and two products are involved . There are
different types of bi bi reaction –
 Random Ordered Bi Bi Reaction

In random ordered bi bi reaction either substrates can bind first to the enzyme and either products can
leave first. Regardless of which substrate binds first the reaction goes through an intermediate ternary
complex (E.AX.B),as illustrated.
Example-
 Compulsory Ordered Bi Bi reaction

In compulsory bi bi reaction,one substrate say A, must bind to the enzyme before the other substrate
(B) can bind. As with random ordered reactions the mechanism proceed through formation of a ternary
intermediate. In this reaction scheme is as follows -
Example- Lactate dehydrogenase
Double Displacement or Ping Pong reaction

 In a ping pong reaction,lacking a ternary complex,the pathway has a transient second form of
enzyme,F .This is form in which a group has been transferred from the first substrate A to create a
transient covalent attachment to the enzyme.
 Such reactions are often called double displacement reactions, as a group is transferred from
first substrate A to the enzyme and then from enzyme to substrate B.
Example-
Steady state kinetic analysis of bisubstrate reactions:

Intersecting line indicate that a

ternary complex is formed in the
reaction
Parallel lines indicate a
ping pong ( double
displacement ) pathway.
Distinguish Between Random And Compulsory
Ordered Mechanisms By Inhibiton Pattern:

 It should be clear that the qualitative form of the double-reciprocal plots makes it easy to
distinguish between a double displacement mechanism and a mechanism involving ternary
complex formation.

 But again, it is not possible to further distinguish between random and compulsory ordered
mechanism.

 If there is an inhibitor that binds to the same site on the enzyme as one of the substrates will slow
the overall forward rate of enzymatic reaction and can allow one to kinetically distinguish between
random and compulsory ordered reaction mechanism.
 SUMMARY:

 We have seen that enzyme reactions involving two substrates and two products can proceed by at least
three distinct mechanisms: random ordered, compulsory ordered,and double – displacement reactions.

 We can distinguish among these mechanisms on the basis of kinetics measurements,product inhibition
studies.

 The vast majority of enzymatic reactions in nature proceed through the utilization of more than one
substrate to yield more than one product.
 Reference:
 Robert A. Copeland, Enzymes “A practical introduction to,
Structure,Mechanism and Data Analysis” ,second edition , (350-357)
 David L. Nelson, Michael M.cox, Lehninger “Principles of Biochemistry”,
seventh edition,( 207- 211)

 https://en.m.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/sequential_reactions