KINETICS OF ANALOG INHIBITION 249 



(B) The analog may be bound very tightly to the enzyme, in which case 

 the order of addition of the substrate and the analog may be of great im- 

 portance. If the analog is added to the enzyme and the mixture is incubated 

 before the substrate is added, the inhibition may be very marked and the 

 substrate may be unable to displace the inhibitor from the enzyme in a 

 reasonable time. On the other hand, if both substrate and analog are added 

 together, the inhibition may be very low initially but progress slowly, due 

 to the relatively small fraction of the active centers available for reaction 

 with the inhibitor. In either case secondary changes in the enzyme may 

 occur and complicate the kinetics (Chapter 1-12). The basic problem in 

 the interpretation of such inhibitions is the inability experimentally to 

 achieve satisfactory equilibrium conditions, and it must be stressed that 

 the usual inhibition equations and plotting procedures apply only to equi- 

 librium conditions. There has been confusion between noncompetitive and 

 irreversible inhibitions. The arsenicals, the mercurials, and iodoacetate, 

 for example, are often thought to inhibit succinate dehydrogenase non- 

 competitively, and yet succinate and these inhibitors react at the same site 

 on the enzyme, as shown by the protection afforded by the presence of 

 succinate when it is added with the inhibitors. These inhibitions are indeed 

 competitive under certain conditions and during specific time intervals, 

 but once the inhibition has been established it is difficult to demonstrate 

 a competitive effect. Several examples of this situation using analog inhi- 

 bitors will be encountered in this chapter. 



(C) The concentration of free inhibitor may be depleted due to its com- 

 bination with the enzyme or other materials and one is then dealing with 

 a mutual depletion system (Chapter 1-3). The quantitative aspects of com- 

 petition may be modified quite markedly in such cases. This behavior must 

 be looked for particularly when one is working with very potent analog 

 inhibitors. 



(D) An irreversible or semi-irreversible change in the configuration or 

 properties of the active center may occur following reaction with the inhibi- 

 tor, so that even after dissociation of the inhibitor from the enzyme the 

 affinity of the enzyme for the substrate is altered. The active centers of 

 certain enzymes appear to be flexible and adapt in some way to the in- 

 teracting molecules, and it is possible that such a change would not be readily 

 reversible. The active center, or at least the immediately adjacent region, 

 of the penicillinase of Bacilltis cerevs is altered by combination with the 

 competitive analog of benzylpenicillin, in that there is a marked increase 

 in the sensitivity to iodination, and this is prevented by the presence of 

 substrate (Citri and Garber, 1961). Although this alteration is presumably 

 reversible, since the ability to hydrolyze benzylpenicillin after removal of 

 the inhibitor is unimpaired, it is easy to imagine changes only slowly re- 

 versible. 



