Transient-state kinetics of L-glutamate dehydrogenase: mechanism of alpha-ketoglutarate inhibition in the burst phase.
Stopped-flow studies of the initial burst of NADPH production accompanying the oxidative deamination of L-glutamate by L-glutamate dehydrogenase and NADP+ were performed in the presence of alpha-ketoglutarate, a product of the reaction. Both binary enzyme-alpha-ketoglutarate and ternary enzyme--NADP+-alpha-ketoglutarate complexes are inhibitory in the burst presence of the enzyme-catalyzed reaction. Order-of-addition experiments show the binary complex to form rapidly, in the 3 ms dead time of the stopped-flow instrument. There is a distinct lag, however, in the achievement of the full ternary complex inhibitory effect unless the enzyme is preincubated with both NADP+ and alpha-ketoglutarate prior to initiation of the catalytic reaction with L-glutamate. The formation of an inhibitory enzyme--NADP+-alpha-ketoglutarate complex appears to be sufficiently slow to give a delayed kinetic response when alpha-ketoglutarate is added to the reaction system.
D000818:Animals; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors*; D007656:Ketoglutaric Acids / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008433:Mathematics; D011485:Protein Binding; D013997:Time Factors
D007656:Ketoglutaric Acids; D005969:Glutamate Dehydrogenase