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Kinetics of the Glyceraldehyde 3-Phosphate Dehydrogenase-catalyzed Hydrolysis of p-Nitrophenyl Acetate

From the ¹ From the Department of Chemistry, Indiana University, Bloomington, Indiana 47401

The kinetics of hydrolysis of p-nitrophenyl acetate by rabbit muscle glyceraldehyde 3-phosphate dehydrogenase has been investigated in aqueous solution at 25° in the pH range 6 to 9. Steady state kinetics was examined under conditions in which substrate is present in great excess over enzyme; pre-steady state kinetics was examined under similar conditions and also when substrate and enzyme are present in approximately equal concentrations. As previously observed, the p-nitrophenolate-time curves were biphasic, exhibiting a rapid exponential reaction, reflecting principally the rate of enzyme acylation, followed by a zero order reaction, reflecting principally the rate of enzyme deacylation. The pH-rate profiles for both acylation and deacylation reactions were carefully examined and found to be similar. In each case, the rate constants increase linearly with increasing hydroxide ion concentration below pH 7.5 and then level off with increasing pH in a manner consistent with the ionization of single groups. Thus, kinetically, acylation depends on a basic group, the conjugate acid of which has a pK_a near 8.1, and deacylation depends on a similar group, with pK_a near 8.7. The number of moles of p-nitrophenolate released per mole of enzyme in the exponential phase of the reaction is a function of pH, rising from about 2.5 to 3.0 below pH 8 to 5.5 above pH 8. Under steady state conditions, an extraneous reaction was noted at very high substrate concentrations; that is, the rate law under these conditions has the form v = k₃E₀ + k_iiiE₀S₀. For enzyme acylation with p-nitrophenyl acetate, the magnitude and pH dependence of the rate constants are consistent with unaided attack of a crucial sulfhydryl anion on the ester. In contrast, for deacylation, the magnitude and pH dependence of rate constants require the involvement of a second catalytic entity of the enzyme.

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