l‐Glutamate Dehydrogenase. The Catalytic Properties of the Mercurial‐Activated Enzyme

Glutamate dehydrogenase can be modified by reaction of cysteine residue 319 with p ‐chloromercuribenzoate or methyl mercuric chloride. The catalytic properties of these modified enzymes have been investigated. Both mercurial enzymes are able to catalyse glutamate oxidation, but their apparent affinities for substrate are decreased, particularly at those sites involved in the inhibition process at high glutamate concentration. Like the native enzyme, both mercurial enzymes exhibit downward curvature in Lineweaver‐Burk plots of reaction velocity against either substrate or coenzyme concentration. Study of the pH‐dependence of the first prestationary step of glutamate oxidation demonstrates that the maximum rate constant (120 s −1 at 15°C) occurs with the ionization of a charged group of p K a 8.2, possibly involved in the hydride transfer. The p K a of this charged group is shifted 0.7 in the case of the methyl mercurial enzyme (p K a 8.9 and k app = 100 s −1 at 15 °C), and 0.2 in the case of p ‐mercuribenzoate enzyme (p K a 8.4 and k max app = 30 s −1 ). The modifications with mercurial reagents also induce a change in the pH‐dependence of the steady‐state velocity. The maximum velocity is 2 s −1 between pH 7.5 and 8.0 for the native enzyme, 4 s −1 above pH 8.8 for the methyl mercurial enzyme and 2 s −1 above pH 8.5 for the p ‐mercuribenzoate enzyme. This capacity of methyl mercuric chloride to increase the rate of enzyme turnover has been correlated with an increase in the rate of dissociation of products. The mercurial‐induced perturbations of the rate constants of the prestationary steps and stationary state, and of their pH‐dependence, modify the time resolution of the absorbance and fluorescence during the glutamate oxidation in a manner similar to those induced in the native enzyme by the binding of ADP.