ANALOGS OF NICOTINAMIDE 487 



amide {K^s usually between 20 and 100 mM). The NADase from Neuro- 

 spora crassa is quite resistant to nicotinamide (Kaplan et al., 1951). 



The inhibition by nicotinamide is competitive with respect to NAD for 

 the weakly inhibited NADases of rabbit erythrocytes (Alivisatos et al., 1956; 

 Hofmann, 1955), lupine seedlings (Hasse and Schleyer, 1961), and Neuro- 

 spora. However, the inhibition of the sensitive mammalian NADases from 

 brain and spleen is noncompetitive and the elucidation of the mechanism 

 by Zatman et al. (1953) has provided important information on NAD me- 

 tabolism and its inhibition by a variety of agents. The binding of nicotin- 

 amide to the enzyme is, however, readily reversible upon dilution or dialysis. 

 The following reaction mechanism was suggested as a working hypothesis: 



+NRPPRA + enzyme ^ enzyme - +RPPRA + N 



l+H.O 

 enzyme + RPPRA + H+ 



An intermediate enzyme complex which is subsequently hydrolyzed is as- 

 sumed. The hydrolysis is irreversible and N + RPPRA will not form NAD. 

 The inhibition by nicotinamide is thus a competition with water for the 

 enzyme-+RPPRA complex. This complex is not a Michaelis-Menten ES 

 complex but a covalent-linked compound in which the energy of the ni- 

 cotinamide — riboside bond is conserved. The free energy for the hydrolysis 

 of this bond is — 8.2 kcal/mole, and its conservation in the complex is very 

 important for the exchange reactions catalyzed by this enzyme. If this 

 mechanism is valid, one should observe exchange between free nicotinamide 

 and the nicotinamide in NAD, and this was demonstrated by using nicotin- 

 amide-C^*. These NADases might be considered as transglycosidases and 

 able to transfer the RPPRA group to compounds structurally related to 

 nicotinamide to form NAD analogs (Zatman et al., 1954 a). The NADases 

 which are weakly inhibited do not operate by such a mechanism and do 

 not catalyze exchange reactions. 



Another enzyme which is nicotinamide-sensitive and catalyzes a similar 

 exchange reaction is the nicotinamide riboside phosphorylase of human 

 erythrocytes (Grossman and Kaplan, 1958 a, b). l/w-l/(S) plots showed the 

 inhibition to be uncompetitive, which is usually interpreted as a combina- 

 tion of the inhibitor with the ES complex, but in this case is perhaps due 

 to the transfer nature of the reaction. In the scheme: 



ERN v=^ E + RN 



'> 



ER '^ 



ERP — > E + RP 



where nicotinamide riboside is the substrate and — d(Rl^)ldt = /-(ERP), it 

 is seen that nicotinamide will slow the reaction by shifting the equilibrium 



