Intermediates in the reaction catalyzed by glutamate dehydrogenase.

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By studying the glutamate dehydrogenase catalyzed oxidation of glutamic acid under conditions where the concentration of enzyme exceeded that of NAD, it was possible to evaluate directly the first‐order rate constant (0.73 sec−1) for the decay of an intermediate associated with the burst in absorbance, which is seen at 340 nm in the pre‐steady state phase when the NAD concentration exceeded that of enzyme. A study of the wavelength dependence of the absorbance vs time plots when [enzyme] > [NAD] indicated that the intermediate associated with the burst is isosbestic with the ternary abortive complex E · Glu · NADH. This information made it possible to estimate the total amount of NADH (π) formed in the initial rapid phase when [NAD] > [enzyme].A study of the dependence of the pre‐steady‐state kinetics on [NAD] revealed that between 27 and 1000 μM NAD, π was invariant at 0.4 μmol NADH formed in the burst phase per polypeptide chain. However, the velocity of the phase immediately following the burst phase increased between 27 and 1000 μM NAD, and the pseudo‐first‐order rate constant for the burst phase increased between 30 and 300 μM NAD and decreased between 300 and 1000 μM NAD. These results were taken to mean that all the NAD sites on glutamate dehydrogenase are not catalytically equivalent and that binding of NAD to sites which are not catalytically active causes a decrease in the rate of the burst phase and an increase in the rate of the phase following the burst.Below 1000 μM glutamate, a pronounced lag in the onset of the burst phase is seen. This lag suggests that at low glutamate concentration binding of NAD and/or glutamate to the enzyme becomes slower than reduction of E · Glu · NAD.