90 3. KINETICS OF ENZYME INHIBITION 



equation for the uninhibited reaction 2-71 and a rather complex expression 

 for the inhibition may be obtained. This was applied to the inhibition by 

 glycine of the hydrolysis of glycerophosphate by alkaline phosphatase using 

 zinc as the activator, and the constants were evaluated from the appro- 

 priate reciprocal plots. This treatment assumed that the concentration of 

 free activator is negligible and that the activator is mainly present combin- 

 ed with substrate and inhibitor; if the affinity of the enzyme for the acti- 

 vator is high or the concentration of activator is sufficient to form EA com- 

 plex, the expressions become much more complex. 



An interesting example of complicated inhibition kinetics involving a 

 coenzyme is provided by D-amino acid oxidase (Yagi et al., 1959a, b). 

 Certain inhibitors form complexes with flavin adenine dinucleotide (FAD) 

 (phenol, salicylate, m-aminophenol, and chlortetracycline). The complex 

 between FAD and chlortetracycline was demonstrated by the quenching 

 of the flavin fluorescence and the shift in flavin absorption spectrum. Indeed, 

 it was claimed that chlortetracycline has a single inhibitory mechanism on 

 this enzyme, the formation of the complex with the coenzyme. However, 

 the phenols and certain other compounds (aniline, j?-aminobenzoate, etc.) 

 can compete with FAD for the coenzyme site. Thus phenol, for example, 

 inhibits by two mechanisms related to the coenzyme. Finally, some of these 

 substances can compete with the substrate. Salicylate inhibits by all three 

 mechanisms and presents a good example of a situation that is ])robably 

 more common than generally believed. It is too often assumed in interpret- 

 ing the experimental data that a single mechanism of inhibition is operative 

 and many of the deviations from the expected behavior may be due to 

 multiple actions. 



THE ENZYME COMBINES WITH MORE THAN ONE 

 MOLECULE OF INHIBITOR 



It has been occasionally assumed that if n molecules of inhibitor react 

 with the enzyme, noncompetitive inhibition is expressed by: 



(I)" 



^ ' (3-81) 



(I)" + K/ 



and competitive inhibition by a similar equation with K/ multiplied by 

 1 + [(S)/^^]. However, it is doubtful if this type of equation ever corre- 

 sponds to an actual situation, since it assumes either that no intermediate 

 complexes (EI, EI2 to EI„_J are formed or, if they are formed, they do 

 not modify the enzymic activity (i.e., the addition of the >ith inhibitor mol- 

 ecule suddenly reduces the activity to zero). Furthermore, it is evident 

 that K/ is not a simple dissociation constant, since it equals (E)(I)"/(EI,j); 



