ANALOGS OF NICOTINAMIDE 513 



form ADP and ATP leads to about 1.2 kcal/mole increase in binding energy. 

 The addition of the 2'-phosphate to form NADP from NAD increases the 

 binding markedly and changes the nature of the inhibition. It is rather 

 strange that the addition of nicotinamide riboside to ADP lowers the bind- 

 ing energy about 1 kcal/mole. 



Williams (1952) found that malate dehydrogenase is inhibited by adenine, 

 adenosine, and ATP. From this observation he concluded that such nor- 

 mally occurring substances may well affect dehydrogenases and other en- 

 zymes in the cell. His work stemmed from the report of Raska (1946) that 

 administration of 300-500 mg/day adenine to dogs on normal diets leads 

 to the development of multiple avitaminosis after 10-20 days; signs of ni- 

 cotinate deficiency, such as black tongue, were noted. The many more data 

 now available serve to strengthen Williams' conclusion, since even more 

 potent inhibitors have been reported. There has been much speculation 

 concerning the regulation of oxidative reactions by adenine nucleotides me- 

 diated through coupled phosphorylation. It is quite possible that other more 

 direct effects on dehydrogenases occur, both in the cell (particularly in the 

 compartmentalized mitochondria) and in experimental enzyme prepara- 

 tions where the concentrations of added nucleotides are often high enough 

 to inhibit appreciably. From Table 2-31 we see that five enzymes are inhi- 

 bited from 22% to 50% by ATP at concentrations from 1 mM to 3.4 mM; 

 ATP is commonly added to mitochondrial preparations at these or higher 

 concentrations. An experimental survey of dehydrogenase inhibitions by 

 nucleotides would be valuable. Chen and Plant (1963), on the basis of the 

 fairly potent inhibitions exerted by certain nucleotides on the NAD-link- 

 ed isocitrate dehydrogenase (Table 2-31), felt that some regidation of cycle 

 activity may be exerted, and if such does occur it would be a very impor- 

 tant factor in understanding not only the effects of nucleotide analogs but 

 also of many inhibitors which either primarily or secondarily alter the levels 

 of cellular or mitochondrial nucleotides. Another interesting point has been 

 brought out by Dalziel (1962) in connection with possible impurities in 

 preparations of the coenzymes. Although one might expect in many cases 

 an insignificant inhibitory effect of certain analogs because they have been 

 shown to bind less tightly than the normal coenzyme to the apoenzyme, 

 Dalziel correctly states that it is the relative values of K^ and K^^ which 

 are important, and K,,^ can be much higher than K^.. He calculated that 

 the presence of an analog of NADH as a 3% impurity can produce as much 

 as 70% inhibition of liver alcohol dehydrogenase if the analog and NADH 

 have the same affinity for the apoenzyme. 



An NAD(P) analog which would bind to the NAD(P) site on dehydroge- 

 nases and then react chemically with some group at the site might well 

 be of some value in labeling these sites. Such an analog was investigated 

 by van Eys et al. (1962) on the basis that thiazole rings often open at al- 



