MECHANISM OF ACTION OF CALF SPLEEN NAD+ GLYCOHYDROLASE

The hydrolysis of NAD+ analogs bearing different substituents in the pyridinium moiety, catalyzed by solubilized calf spleen NAD+ glycohydrolase, was studied. The enzyme was specific for analogs possessing a carbonyl function at position C-3. The observed maximal rates showed no simple dependence either on the leaving ability of the parent pyridines or on the observed binding energies. The analogs without carbonyl substituents were not found to be hydrolyzed under our experimental conditions; and, compared to the substrates, they all presented much higher affinities for the active site, which could not be related to specific interactions. These results indicate that the rate-limiting step of the NAD+ hydrolysis, which is the formation of an enzyme-ADP-ribosyl intermediate, is probably more complex than a simple chemical step, i.e., pyridinium-ribose bond breakage. At a molecular level we favor a catalytic mechanism which, through nonbonded interactions between the substrate and the active site of the enzyme, results in the destabilization of the pyridinium-ribose bond, i.e., unimolecular decomposition involving an oxocarbonium ion intermediate. © 1979.