INHIBITION OF ENZYMES 771 



Types of Inhibition Observed with the Mercurials 



The concentration-inhibition curves for mercurials are generally sigmoid 

 and rather steep, as would be expected of inhibitors combining tightly with 

 enzyme groups. Indeed, when such curves are fairly flat, encompassing sev- 

 eral pi units, one has the right to question if the inhibition is related to 

 mercaptide formation, although it may well be. It should be emphasized 

 that adequate kinetic studies can be made only in preparations of pure 

 enzymes. The presence of impurities may distort the entire picture and the 

 kinetics of inhibition. 



One may classify the inhibitions classically into competitive, noncompet- 

 itive, uncompetitive, and mixed types, but the proper plotting procedures 

 have seldom been used so that in the majority of cases we have little or no 

 information. However, sufficient has been done to show that all these types 

 of inhibition occur (Table 7-8). Competitive behavior has been observed 

 in a surprisingly large number of instances. This is surprising at first if one 

 assumes reaction with SH groups to be the primary mechanism of inhibi- 

 tion, because the tightness of the binding might be considered to prevent 

 the exhibition of competition. Actually, most inhibitions by mercurials are 

 probably competitive — either with substrate, coenzyme, or cofactor — in 

 the fundamental sense of the word, but it is often difficult to demonstrate 

 this by the usual analytical techniques which assume equilibrium conditions. 

 It is easier to show that the presence of the substrate, coenzyme, or cofactor 

 slows the development of the inhibition, although the equilibrium inhibition 

 may not be detectably different (page 778). Formally competitive behavior 

 might be expected to occur in the following circumstances. (1) The inhibitor 

 acts by a non-SH reaction; the organic mercurials particularly possess group- 

 ings capable of interacting with active sites independently of the Hg atom, 

 and such might be involved, for example, in the inhibition of D-amino acid 

 oxidase by p-MB, the benzoate structure being of primary importance. (2) 

 The binding of the mercurial to the SH groups may for some reason be 

 weaker than usual and of a comparable magnitude to the affinity for the 

 substrate. (3) The mercurial is bound much more tightly than the substrate 

 but measurements are made before equilibrium is reached, as in the exper- 

 iments showing protection of the enzyme by the substrate; when the inhi- 

 bitions are determined soon after adding the mercurial in the presence of 

 variable concentrations of the substrate, the data may provide formally 

 competitive plots. One would expect this third explanation in certain exam- 

 ples given in Table 7-8, e.g., carbonic anhydrase, where KJK,,^ = 3.87 X 10^' 

 for ??-MB (Chiba et al., 1954 b). In the case of homogentisate oxidase, p-MB 

 and MM inhibit competitively with respect to Fe++ but noncompetitively 

 with respect to homogentisate, the mercurials being bound roughly 40-100 

 times as tightly as the Fe++ (Flamm and Crandall, 1963). Here, and in 

 other instances where metal ion cof actors are involved, both cofactor and 



