Calf‐Spleen Nicotinamide‐Adenine Dinucleotide Glycohydrolase Kinetic Mechanism

Steady‐state analysis of product inhibition indicates that the most probable minimum kinetic mechanism for soluble calf spleen NAD glycohydrolase is ping‐pong bi‐bi, reducing to an ordered uni‐bi for hydrolysis alone. In the hydrolysis of NAD nicotinamide is released first. Isotope exchange, transglycosidation reaction and nucleophilic competition with methanol show that the formation of the intermediary enzyme · adenosine‐diphosphoribosyl complex (E‐ADP‐Rib) is rate limiting. The NAD hydrolysis is subject to inhibition by excess of substrate, which is markedly accentuated by increasing concentrations of nicotinamide. Kinetic analysis of this behaviour is in favour of the formation of an apparent dead‐end complex of NAD with the Michaelis complex. The reactivies of methanol and nicotinamide compared to water with the intermediate E‐ADP‐Rib suggest the occurrence of an intermediary oxocarbonium ion in the reaction catalyzed by NAD glycohydrolase.