506 2. ANALOGS OF ENZYME REACTION COMPONENTS 



exerted in cellular metabolism, and (3) to find useful inhibitors that may 

 specifically inhibit particular reactions in complex systems. Some of the 

 results on different types of enzyme involving NAD or NADP are summa- 

 rized in Table 2-31. It is unfortunate that in very few instances have the 

 types of inhibition been determined and it is seldom possible to calculate 

 accurately the K/s or even relative K/s, from which interesting binding 

 energy information might be obtained. 



One may first ask: Does the inhibitory activity generally increase as ri- 

 bose and phosphate groups are added? The answer is roughly in the ajBfir- 

 mative for NAD kinase, NADH pyrophosphatase, NADH oxidase, NAD : 

 NADP transhydrogenase, alcohol dehydrogenase, and malate dehydroge- 

 nase, but in a few enzymes there appears to be no definite trend, while in 

 some the addition of a group may reduce the binding. The addition of a 

 phosphate to adenosine to form 5'-adenylate (AR — »■ 5'-ARP) leads to only 

 0.1 kcal/mole extra binding to the alcohol dehydrogenase and 0.4 kcal/mole 

 to the NAD kinase, but an increased binding of over 2.4 kcal/mole for the 

 NADH pyrophosphatase. The further addition of a phosphate to form ADP 

 increases the binding approximately 0.7 kcal/mole for the NAD kinase, 

 1.1 kcal/mole for alcohol dehydrogenase, and 0.6 kcal/mole for the NAD : 

 NADP transhydrogenase, whereas the binding to NADH oxidase or NADH 

 pyrophosphatase is unchanged or slightly reduced. Addition of another phos- 

 phate to form ATP leads to increased binding only for the liver NADH 

 oxidase. Addition of a ribose to ADP to form ARPPR has no effect for 

 NAD kinase but increases the binding around 0.7 kcal/mole with NADH 

 pyrophosphatase. Final addition of nicotinamide to ARPPR to form NAD 

 increases the binding around 1.9 kcal/mole for NAD kinase and NADPH- 

 glutathione reductase, whereas a reduction of 1.9 kcal/mole in the binding 

 to NADH pyrophosphatase is observed. Addition of nicotinamide to 2'-P- 

 ARPPR to form NADP leads to a 2.3 kcal/mole increase in binding for the 

 NADPH-glutathione reductase and to very little change for the NADP- 

 cytochrome c reductase. The marked variation in behavior between en- 

 zymes and the uncertainty in the accuracy of the energy values make it im- 

 possible to draw definite conclusions or formulate rules for these inhibitions. 

 It appears that all the components of the NAD and NADP molecules can 

 participate in the binding, although not all of them need function for a 

 particular enzyme. The rather marked inhibition occasionally exerted by 

 NADH on NAD reactions, or by NAD(P) on NAD(P)H reactions, indicates 

 not only the specificity of these enzymes but points to a somewhat different 

 orientation of the oxidized and reduced forms on the enzymes. 



A more interesting correlation emerges when one considers the variation 

 of inhibitory potency with the position of phosphate groups on the adenyl 

 ribose. In NAD the 5-position is phosphorylated and enzymes involving 

 NAD are more readily inhibited by 5'-AMP than by 2'- or 3'-AMP (NAD 



