NICOTINAMIDE NUCLEOTIDE ENZYMES 



111 



The next problem was to determine what type of linkage existed 

 between nicotinamide and the rest of the molecule and what 

 changes were brought about in the ultraviolet absorption by reduc- 

 tion of the coenzymes to the dihydro form. This question was an- 

 swered by the extensive experiments of Karrer and his co-workers. 

 In the first place, the three functional centers of the nicotinamide 

 molecule had to be taken into consideration. "Model compounds" 

 were therefore prepared which were substituted in these positions 

 with simple organic groups. Among these compounds may be men- 

 tioned the following typical representatives: nicotinamide iodo- 

 methylate (I), nicotinic acid ethylimido ether (II), and monomethyl 

 nicotinamide (III). 



FORMULA 1 





CONH2 



OC2H5 



^, 



■CONH-CH, 



N' 



(I) 



(11) 



(HI) 



Of these compounds only the nicotinamide iodomethylate exhib- 

 ited properties similar to the coenzymes. Like the coenzymes it is 

 reducible by hydrosulfite (see formula 2), and the absorption maxi- 

 mum of its dihydro product is 360 m[jL (340 m[x is the typical ab- 

 sorption maximum of the dihydrocoenzymes), which disappears 

 when the solution is acidified, as does that of the dihydrocoenzymes. 



FORMULA 2 



CONH; 



+ 2Y^p 



CONH. 



+ 2NaHS03 

 + HI 



These results indicated strongly that in the codehydrogenases the 

 nicotinamide is bound as a quaternary pyridinium base. The ex- 

 perimental data accumulated in testing this working hypothesis soon 

 established its validity. It was already known that quaternary pyri- 



