Kinetic studies of dogfish liver glutamate dehydrogenase.
Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.
D000641:Ammonia / Q000378:metabolism; D000818:Animals; D004284:Dogfish / Q000378:metabolism*; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D012754:Sharks / Q000378:metabolism*
D005971:Glutamates; D007656:Ketoglutaric Acids; D009243:NAD; D009249:NADP; D000641:Ammonia; D005969:Glutamate Dehydrogenase