INHIBITION OF ENZYMES 51 



Interpretation of Partial Inhibition 



If the carboxymethylation of a group at the active center blocks off a 

 substrate or coenzyme, or otherwise prevents catalysis, the enzyme would 

 be expected to be completely inhibited if the iodoacetate were in excess 

 and sufficient time given for reaction. In other words, under these assump- 

 tions, enzymes should be inhibited in an all-or-none fashion; i.e., either they 

 react or they do not, and in the former case they would be completely inhib- 

 ited. However, most of the results published are partial inhibitions. Partial 

 inhibition may mean (a) insufficient time has been allowed for the reaction, 

 (b) the iodoacetate has been used up, in reaction either with the enzyme 

 or with other substances in the preparation, or (c) the fully reacted enzyrne 

 is not completely inhibited. Most of the results are probably explained by 

 the insufficient time factor, especially in purified preparations in which the 

 iodoacetate is likely to be in excess. There appear to be some cases in which 

 a maximal inhibition is reached in the presence of adequate amounts of 

 inhibitor, and here we must visualize a reaction of iodoacetate with a vicinal 

 SH group, this reducing the affinity of the substrate or coenzymxC for the 

 enzyme, or slowing the breakdown of the ES complex, but not completely. 

 It is usually not easy in a particular instance to determine the cause of 

 partial inhibition. For example, in Fig. 1-6, one does not know if 1 mM 

 iodoacetate will eventually inhibit completely, although it does not appear 

 that it will. However, this was done in a homogenate containing a number 

 of components capable of reacting with iodoacetate, so perhaps the inhibitor 

 is depleted at this concentration. The near-complete inhibitions observed at 

 higher concentrations could be taken as evidence that this is the case, but 

 groups other than SH may be attacked at these high concentrations. One 

 is also somewhat troubled by the shapes of most of the inhibition-time 

 curves, inasmuch as usually there is an initial relatively rapid reaction, which 

 does not inhibit the enzyme completely, followed by a slower reaction or 

 reactions. Are these slower phases due to reaction with less reactive groups 

 on already reacted enzyme, or to secondary inactivation unrelated to further 

 carboxymethylation ? 



Summary of Enzyme Inhibition 



Iodoacetate and iodoacetamide at concentrations below 5 mM and in the 

 physiological pH range probably react with enzyme SH groups much more 

 rapidly than with other groups, although there is always the possibility 

 that a particular amino acid residue is in a specially reactive state. The 

 inhibitions develop slowly and seldom reach completion before 30-60 min. 

 In most cases the enzyme is protected by the presence of substrate or co- 

 enzyme. Inhibition is often irreversible but in some instances partial or 

 complete reactivation may be achieved by dialysis or treatment with thiols. 



