Biochemistry Online: An Approach Based on Chemical Logic

Biochemistry Online




Last Update:  04/11/16

Learning Goals/Objectives for Chapter 6D:  After class and this reading, students will be able to

  • draw Cleland chemical reaction diagrams showing enzyme, substrate, and product interactions for multisubstrate and multiproduct sequential and ping-pong enzyme-catalyzed reactions;
  • draw double reciprocal 1/v vs 1/A plots at different fixed B concentrations for sequential and ping-pong multisubstrate reactions;
  • draw v vs S graphs in the presence and absence of allosteric inhibitors and activators for multi-subunit enzymes that display sigmoidal cooperative behavior (K systems) conforming to the MWC model;
  • differentiate between K and V systems for allosterically regulated enzymes using the MWC model and explain shifts in graphs of v vs S in the presence of allosteric effectors

D1.  Multi-Substrate Sequential Mechanisms

In reality, many enzymes have more than one substrate (A, B) and more than one product (P, Q).  For example, the enzyme alcohol dehydrogenase catalyzes the oxidation of ethanol with NAD (a biological oxidizing agent) to form acetaldehyde and NADH.   How do you do enzymes kinetics on these more complicated systems?  The answer is fairly straightforward.  You keep one of the substrates (B, for example) fixed, and vary the other substrate (A) and obtain a series of hyperbolic plots of vo vs A at different fixed B concentrations.  This would give a series of linear 1/v vs 1/A double-reciprocal plots (Lineweaver-Burk plots) as well.  The pattern of Lineweaver-Burk plots depends on how the reactants and products interact with the enzyme.

Sequential Mechanism:  In this mechanism, both substrates must bind to the enzyme before any products are made and released.  The substrates might bind to the enzyme in a random fashion (A first then B or vice-versa) or in an ordered fashion (A first followed by B).  An abbreviated notation  scheme developed by W.W. Cleland is shown below for the sequential random and sequential ordered mechanisms.  For both mechanisms, Lineweaver-Burk plots at varying A and different fixed values of B give a series of intersecting lines.  Derivative curves can be solved to obtain appropriate kinetic constants.

sequential.GIF (10382 bytes)


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