502 



2. ANALOGS OF ENZYME EEACTION COMPONENTS 



are more reliable than in the tabulation above. The K^^^ for NAD is 0.00428 

 mM (relative —AF would be 7.63 kcal/mole) so that none of these inhi- 

 bitors are bound nearly so tightly. Substitution in the 3-position of the pyri- 



coo 



CONH, 



Nicotin- 

 amide 



SOpNH, 



Pyridine-3- 

 sulfonamide 



CONHNH, 



Isonicotinyl 

 hydrazide 



OH 



CHO 



COO 



COO 



3 -Hydroxy - 

 pyridine 



Salicylate 



CHoOH 



CH,OH 



dine ring is necessary for significant inhibition, but the nature of this group 

 can vary considerably and certainly no marked electrostatic interaction is 

 involved. The pyridine N would not seem to be of much importance in the 

 binding, since benzamide is about as inhibitory as nicotinamide, and ben- 

 zenesulfonate almost as potent as pyridine-3-sulfonate, and yet A"-methyl- 

 ation (to form trigonelline) abolishes the inhibition. The extra 2.1 kcal/mole 

 provided by the 3-hydroxy group suggests the possibility of hydrogen bond- 

 ing to the enzyme from this position, but dipolar and dispersion interactions 

 could also account for this. It may be noticed that the results with alcohol 

 dehydrogenase (Table 2-30) are in certain respects different than those 

 with glucose dehydrogenase. In this connection one must remember that 

 the ionization constants of these analogs should be considered, and it may 

 be that the major effect of the substituent groups is by modification of the 

 p^a of the pyridine N. Until these problems have been treated quantita- 

 tively, it is impossible to evaluate accurately the relationship between 

 structure and inhibitory activity. In any event, it is clear that the major 

 binding energy of NAD is contributed by the adenine nucleotide portion 

 of the molecule, so that pyridine derivatives might not be expected to be 

 potent inhibitors of dehydrogenases. The relatively strong inhibitions pro- 

 duced by pyridoxal and 4-pyridoxate may be significant for cellular meta- 

 bolic regulation and further study of the effects of these substances on 

 various dehydrogenases is probably warranted. 



Nicotinamide has been frequently used in homogenates to inhibit the 

 splitting of NAD by NADases, as discussed above, and often at concentra- 



