REVERSAL BY A SUBSTAXCE BINDING THE INHIBITOR 625 



the inhibitor, it is necessary to evaluate critically the information one may 

 obtain from such experiments. There are two minor things one may possibly 

 learn from reversal studies: (1) an estimate of the relative affinities of 

 the enzyme and reversor for the inhibitor, and if K^ is known, K^ may be 

 calculated approximately; and (2) if complete reversal occurs, no secondary 

 reactions leading to inactivation of the enzyme have taken place. Xn esti- 

 mate of K^ may be made more readily from the usual graphical procedures 

 and a test for inactivation can be done with less complexity by dialysis. 

 This is not the information that has generally been looked for in such work. 

 Instead, conclusions as to the mechanism of inhibition have often been 

 drawn. There is no relationship between the way in which the inhibitor re- 

 duces the activity of the enzyme and the reversibility. From the rate at 

 which an inhibitor can be removed from an enzyme, or from the degree 

 to which the inhibition may be reduced by the addition of an inhibitor- 

 binding substance, one can tell nothing as to the mechanism of the primary 

 inhibition. It has been stated many times that if the inhibition produced 

 by a sulfhydryl agent is reversed by a sulfhydryl compound (such as 

 cysteine, glutathione, or dimercaprol), this is evidence for the reaction of 

 the inhibitor with enzyme sulfhydryl groups as the mechanism of the in- 

 hibition. Such a conclusion is not valid and it should be so obviously not 

 valid that no discussion of the problem is warranted. 



Let us now consider some i^roblems in the interpretation of partial or 

 complete irreversibility. Failure to restore the total enzyme activity is 

 not certain proof of either an irreversible binding of the inhibitor to the 

 enzyme or of secondary reactions leading to irreversible changes in the en- 

 zyme. Homogentisate oxidase is well inhibited with /j-CMB but glutathione 

 will not reactivate the enzyme (Crandall, 1955). However, if Fe"^"^ ions 

 are added with the glutathione, restoration of activity occurs. The p-CMB 

 by reacting with the enzyme has displaced the Fe"^"^ ions necessary for 

 catalytic activity and removal of the p-CMB alone, by any means, will not 

 reverse the inhibition. Ferrous ions alone will also not reactivate because 

 they cannot reach the activator site on the enzyme. But removal of the 

 inhibitor followed by addition of the activator abolishes the inhibition. This 

 phenomenon may be rather common, inasmuch as it is known that inhi- 

 bitors can displace cofactors and coenzymes; the displacement of DPN+ 

 from certain enzymes by the mercurials would be an example. 



Another possible explanation for irreversibility would be an effect of 

 the reversor on the enzyme. If the reversor in any way were an inhibitor 

 or inactivator of the enzyme, it would simultaneously remove the original 

 inhibitor and depress the activity of the enzyme. Reactivation of succin- 

 oxidase inhibited by p-CMB (Slater. 1949) provides an excellent illustration 

 of this. Certain thiols used for reactivation were oxidized in the enzyme 

 preparation (presumably by dissolved oxygen and catalyzed by substances 



