REVERSAL BY A SUBSTANCE BINDING THE INHIBITOR 623 



of the action of a chemical or physical agent that inhibits sulfhydryl groups" 

 (Hargreaves, 1955), are common and misleading. If a series of enzymes is 

 inhibited with the same inhibitor and reactivation is attempted in each 

 case with the same reversor, it might be expected that all degrees of re- 

 versibility would be exhibited because the enzymes would show a wide 

 range of affinities for the inhibitor. The results would depend primarily 

 upon the relative dissociation constants of the inhibitor with enzyme and 

 with reversor. Attempting to reverse an inhibition with a substance whose 

 TpK^ is much lower than the p^^ for the inhibition is as futile as trying to 

 oxidize or reduce a substance with another whose redox potential is inap- 

 propriate. The inhibitions that gave no reversal above probably involve 

 the reaction of sulfhydryl groups as in those cases where complete resto- 

 ration of activity occurred. The different degrees of reversibility indicate 

 most likely the relative affinities of the various enzymes for the mercurial. 



No measurements of the rates of reversal seem to have been reported. 

 In some cases it is surprisingly rapid. The reactivation of p-CMB-inhibited 

 glyceraldehyde-3-phosphate dehydrogenase by cysteine is so rapid that 

 technically it is not possible to measure it (Velick, 1953, 1954). The addi- 

 tion of cysteine in a concentration about a hundred times greater than the 

 p-CMB concentration produced immediate and almost complete reversal 

 (Fig. 13-8). Similar results were obtained with brain hexokinase (Sols and 

 Crane, 1954), although the reversal w^as not as complete. These results 

 show that the reaction of cysteine with js-CMB and the dissociation of 

 p-CMB from the enzyme are reasonably fast, but no information is available 

 as to which reaction is limiting the rate. Also it is not known how these 

 rates of reversal compare with the rates that would be observed in experi- 

 ments where the inhibitor was removed by other means, such as dialysis. 

 If it could be demonstrated in any case that the rate of reversal with a 

 substance binding the inhibitor is greater than the rate found when the 

 inhibitor was removed by physical means, it would indicate strongly that 

 the reversor is able to react with the inhibitor that is bound to the enzyme 

 and remove it more rapidly than would occur with spontaneous dissociation. 

 It would certainly seem to be very unlikely that such a reaction would oc- 

 cur in most cases, due to steric factors alone, but it is possible, and accurate 

 comparison of reversal rates might provide some information on this in- 

 teresting ijoint. Reactions of two substances on the surface of an enzyme 

 commonly occur but usually the reaction does not involve the groups at- 

 taching the substances to the enzyme. 



One factor that may be very important in reducing reversibility is the 

 progressive inactivation of the inhibited enzyme. It has frequently been 

 observed that enzyme inhibitions become increasingly irreversible. In the 

 case of the inhibition of brain hexokinase with p-CMB, which is, under the 

 proper conditions, completely reversible, increasing incubation times with 



