Coenzyme binding to glutamate dehydrogenase. A study by relaxation kinetics.

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1 The binding of NADH and NADPH to ox liver glutamate dehydrogenase have been studied by the temperature-jump technique using fluorescence detection. 2 The rate of combination of NADH with glutamate dehydrogenase is considerably slower than if it were diffusion controlled while the rate constant for dissociation is too fast for this to be a rate-limiting step in the overall catalytic reaction. 3 The binding constant obtained rom the ratio of the rate constants agrees well with a value previously determined by equilibrium dialysis. 4 Comparison of NADH with NADPH strongly suggests that binding to a site common to both coenzymes is being studied and this must be the active site. 5 The inhibitor guanosine 5′-triphosphate and the activator adenosine 5′-diphosphate both affect the association and dissociation rate constants but not sufficiently to account for the observed steady-state inhibition and activation. 6 In the presence of the substrate l-glutamate a second slower relaxation time is seen and is ascribed to the formation of an abortive ternary complex. This step is not observed with the poorer substrate l-alanine. 7 The data enable a model of the relationship between the active site and the polymerising site to be proposed.