INHIBITION OF ENZYMES 799 



or high temperature), and in this respect the problem is no different from 

 that of protein titration (page 762). We are interested here not so much 

 in the number of SH groups on an enzyme, but how these SH groups relate 

 to the catalytic activity and the mechanisms of mercurial inhibition, and 

 thus in following simultaneously the loss of SH groups and the develop- 

 ment of the inhibition as more and more mercurial is added. Let us assume 

 that we have a solution of a pure enzyme and we add to this a solution 

 of p-MB, or other mercurial, so that the molar ratio of mercurial to enzjnne 

 is slowly increased, and further assume that we allow time for the reaction 

 to come to equilibrium. We measure the number of SH groups reacted 

 (spectrophotometrically, polarographically, argentimetrically, or otherwise) 

 and the enzyme activity. There are only a few fundamental relationships 

 between mercaptidization and inhibition that could emerge, and these are 

 illustrated in Fig. 7-19. These are extreme situations, of course, and inter- 

 mediate behavior would more often be expected. Let us consider what each 

 result may mean and how valid certain interpretations may be. 



Case A: The inhibition runs parallel to the SH groups reacted. 



(1 ) The only SH groups that react are at the active center and mercaptide 

 formation abolishes the enzyme activity; titration to 100% inhibition will 

 give the number of SH groups at the active center. 



(2) There are n equireactive SH groups on the enzyme, but only a cer- 

 tain fraction of these is at the active center or involved in the catalysis; 

 the titration will not provide the actual number at the active center. 



(3) The SH group or groups are not at the active center, but reaction 

 of them leads to inactivation of the enzyme by some means; titration will 

 not provide useful information. 



It is impossible to distinguish between these possibilities by simple ti- 

 tration nor can one determine accurately the number of SH groups at the 

 active center. Let us assume that an enzyme has 10 reactive SH groups 

 totally but that complete inhibition occurs when only 3 are reacted. It has 

 sometimes been concluded that 3 SH groups are necessary for the enzyme 

 activity. This is not a valid conclusion. If 1 of the 3 groups were related in 

 some way to the activity, one would obtain the same data. In other words, 

 the number of equivalents of mercurial added, or the number of SH groups 

 reacted, to achieve 100% inhibition does not provide directly the number 

 of SH groups involved in the catalysis. 



Case B: SH groups are reacted but inhibition does not occur. 



The conclusion here is obvious: the reactive SH groups are not involved, 

 directly or indirectly, in the enzyme activity. It is, of course, possible that 

 there are SH groups at the active center, perhaps even functional, but that 

 they do not react with the mercurial under the experimental conditions. 



