Application of linear free-energy relationships to the mechanistic probing of nonenzymatic and NAD+-glycohydrolase-catalyzed hydrolysis of pyridine dinucleotides

Abstract The use of NAD + analogs lacking a carbonyl function at position C-3 of the pyridinium moiety allowed the manipulation of the kinetic mechanism of calf spleen NAD + glycohydrolase so as to render the cleavage of the pyridinium-ribose bond rate limiting. The analogs used in this study are relatively poor substrates of the enzyme. They present an affinity for the active site which is independent of the nature of their substituent ( K i = 10 ± 2 μ m ), suggesting that the specificity of the NAD + glycohydrolase reflects the dynamic steps occurring after the formation of the Michaelis complex. The maximal rates of hydrolysis of the NAD + analogs are very sensitive to the pK a of the departing pyridine; a Bronsted plot ( r = 0.99) gave a β tg = −0.90 (at 37°C). From this plot we could estimate that for NAD + , the specific interaction of the 3-carboxamide group with the active site contributed to the catalysis by decreasing the energy barrier by about 2 kcal mol −1 . We have also studied the nonenzymatic hydrolysis of NAD + and its analogs under conditions (pH-independent hydrolysis) which favor a unimolecular mechanism. In this case a linear Bronsted plot was also found ( r = 0.99) with β tg = −1.11 (at 37°C). Our data indicate that NAD + glycohydrolase catalyzes the chemical cleavage of the pyridinium-ribose bond, over a 10 3 rate difference, according to a single mechanism involving a late transition state in which the scissile bond is broken. The present study strongly supports our previous hypothesis (F. Schuber, P. Travo, and M. Pascal (1979) Bioorg. Chem. 8 , 83) according to which NAD + glycohydrolase catalyses unimolecular decomposition of its substrates with generation of an ADP-ribosyl oxocarbonium ion intermediate which must be stabilized by the active site of the enzyme.