558 12. RATES OF INHIBITION 



reason or another, it is difficult to arrive reliably at equilibrium inhi- 

 bitions. 



Many examples of the protection against enzyme inhibition that is af- 

 forded by the presence of substrate have been reported. In most cases the 

 protection has not been studied kinetically. Two further instances of protec- 

 tion by substrate will be mentioned in order to clarify the concept of com- 

 petition. Yeast a-carboxylase is inhibited by various sulfhydryl reagents 

 and the rate at which this inhibition occurs is lower the greater the pyru- 

 vate concentration (Stoppani et al., 1953). Protection against p-chloro- 

 mercuribenzoate, HgCl.^, Cu++, arsenicals, o-iodosobenzoate, and iodoace- 

 tate was observed. For example, if ^^-chloromercuribenzoate and pyruvate 

 were added together, the inhibition was 48% after 20 min, but the same 

 concentration of the mercurial in the absence of the substrate inhibited 

 90% within 2 min. The substrate tyramine has been shown to reduce the 

 rate of inhibition of monoamine oxidase by iproniazid (Zeller et al., 1955; 

 Davison, 1957). Tyramine at 50 mM almost completely protected the en- 

 zyme against 0.1 mM iproniazid. For both a-carboxylase and mono- 

 amine oxidase, these results provide evidence that the inhibitors react with 

 the substrate site on the enzyme. Yet these inhibitions are not truly com- 

 petitive in the classic sense because when the enzyme is incubated with 

 the inhibitor alone until maximal inhibition is achieved, the addition of 

 substrate, even in high concentrations, will not reduce the inhibition or 

 displace the inhibitor from the enzyme. One must then distinguish between 

 kinetic competition, where the rate of inhibition is reduced by substrate 

 but the final degree of inhibition is not affected, and equilibrium competi- 

 tion, where both the rate and the final inhibition are decreased by sub- 

 strate. In the former case, the usual plotting procedures will indicate non- 

 competitive behavior when equilibrium inhibitions are measured. How- 

 ever, in this connection, it is obvious that when short experimental periods 

 are used, the inhibition may not have reached an equilibrium value and 

 what one is really measuring is kinetic competition. 



Temperature Effects and the Activation Energy of Inhibition 



Changes in the rate of inhibition with temperature may provide infor- 

 mation about the thermodynamic properties of the activated complex 

 formed between the enzyme and the inhibitor on the reaction pathway 

 towards the normal complex: 



E + I^EI*^EI (12-38) 



Knowledge of the energy changes involved during this sequence — and 

 particularly the alterations in these by varying the pH, ionic strength, 

 and other factors — should be interpretable in terms of the physical events 



