SUBSTRATE INHIBITION 121 



These conclusions are supported by the results of Fridovich and Handler 

 (1958) who showed that substrate inhibition is marked when two-electron 

 acceptors are used to measure the enzyme activity, but does not occur 

 when the one-electron acceptor cytochrome c is used. Furthermore, the 

 substrate inhibition is reduced as the pH is raised and the enzyme non- 

 heme iron becomes increasingly susceptible to auto-oxidation as is shown 

 in the tabulation. It is postulated that the active center includes two mol- 



ecules of FAD bridged by a pair of ferric mercaptide groups; the flow 

 of electrons occurs from the substrate to FAD-1 through the iron atoms to 

 FAD-2 and hence to the two-electron acceptor dyes or oxygen. Cytochrome 

 c, on the other hand, probably withdraws electrons from the non-heme 

 iron directly and FAD-2 is not involved. The evidence thus points to the 

 site of substrate inhibition as the FAD-2, which can presumably react 

 with purines as does the FAD-1 site. 



Several examples of substrate inhibition have been followed over a 

 sufficiently wide range of substrate concentration to conclude that they 

 obey type A-B kinetics, i.e., that they conform to an equation of the type: 



V = Vm (4-14) 



"A -M/(S') +(S')/B 



and give rate-pS curves that are symmetrical. It is not possible, however, 

 to deduce the exact mechanism of the inhibition. Typical curves are illus- 

 trated in Figs. 4-6 to 4-9. It is likely that the esterase inhibition is of type 

 A because of the probability that two binding groups are involved in 

 the active complex; if this is so, the species differences in peak position 

 indicate different degrees of binding interference between the substrate 

 molecules. However, in the case of the /5-hydroxysteroid dehydrogenase 

 it is more difficult to visualize type A inhibition because of the large size 

 of the substrate and the nature of the binding, since the evidence from sub- 

 strate specificity points to the relatively high binding energy as being 

 due to the summation of multiple dispersion forces between the steroid and 

 the enzyme surface. For the Ja ctic dehydr ogenase it is possible that type 

 A inhibition occurs and that the second pyruvate molecule is bound mainly 

 by its aliphatic portion because of the 3 kcal/mole difference between the 



