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

D3.  Inhibitors in Multi-substrate Reactions 

Product Inhibition:  Interpretation of kinetic experiments can be complicated by the fact that the reactions can be reversed.  Even if the catalytic conversions of the reverse steps have too high an activation energy to actual proceed, the products, which obviously have some structural resemblance to the reactants could inhibit the enzyme as they could compete with reactants for binding to the enzyme.  In contrast to studying enzyme inhibition using varying concentrations of substrate at different fixed concentrations of inhibitor, the concentration of products produced by an enzyme are constantly increasing over the time course of the reaction.  This suggests one immediate reason that most kinetic parameters are determined by initial rate methods in which the inhibitor-product has not yet build to a sufficient concentration to alter the rate of conversion of substrate to product.     Product inhibition can occur in single substrate reactions as well.

Dead End Inhibition:  How do added inhibitors affect the double reciprocal plots of multisubstrate reactions?  Let's consider a special case of inhibitors call dead-end inhibitors.  These reversible inhibitors bind to a form of the enzyme and inhibit product formation but do not participate in the reaction.  It would be represented on a Cleland diagram as a vertical line coming off the the horizontal line which represent different enzymes forms (E, EA, EAB, E'Q, EP, EQ, etc) that lead to product formation.    A quick inspection of Cleland diagrams lead to two simple rules that helps in the interpretation of double reciprocal plots in the presence of different fixed and nonsaturating concentrations of dead-end inhibitors (I) in multisubstrate reactions (when one substrate S is varied):

  1. Slope changes when: 


  1. Y Intercept changes when: 

The rules predict 1/v vs 1/S plots for simple competitive  inhibition (S and I bind to the same enzyme form, E) and uncompetitive (S binds to E followed by binding of I to ES).  If the slope changes and the y intercept doesn't, that's competitive inhibition.  If the y intercept changes and the slope remains constant, that is uncompetitive inhibition. It works also for mixed inhibition where I binds to E (the same form as S binds to), which changes the slope, AND also binds to EA (a different form of the enzyme than S binds to which is E), which changes the y intercept.

These same rules apply for product inhibition. Consider the rapid equilibrium ordered bibi reaction above when the concentration of the other substrate is around its Kx value:


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