Interaction of inorganic vanadate with glucose-6-phosphate dehydrogenase. Nonenzymic formation of glucose 6-vanadate

Inorganic vanadate (Vi) activates catalysis by glucose-6-phosphate dehydrogenase of the oxidation of glucose by NADP+. As the concentration of Glu-6-P dehydrogenase is increased, the rate of the vanadate-activated glucose oxidation becomes less sensitive to increases in enzyme concentration. The rate of glucose oxidation in the absence of Vi increases linearly with Glu-6-P dehydrogenase concentration. These results are interpreted in terms of nonenzymic formation of glucose 6-vanadate. At high enzyme concentration, vanadate ester formation becomes partially rate-limiting, and extrapolation to infinite Glu-6-P dehydrogenase concentration allows determination of the second order rate constant for formation of the ester. In separate experiments designed to test the proposed mechanism, it was found that Vi, at concentrations at which it strongly activates catalysis by Glu-6-P dehydrogenase of glucose oxidation, has no effect on the rates of oxidation of glucose 6-phosphate or 6-deoxyglucose catalyzed by Glu-6-P dehydrogenase. Sulfate, which is known to activate glucose oxidation and to inhibit glucose 6-phosphate oxidation, strongly activates 6-deoxyglucose oxidation. These experiments show that the 6-hydroxyl group of glucose is essential for the observed activation by Vi and are also consistent with the formation of glucose 6-vanadate. Also, the rate of the sulfate-activated glucose oxidation increases linearly with Glu-6-P dehydrogenase concentration. These results are consistent with the proposed mechanism for sulfate activation which involves sulfate binding to the enzyme (Anderson, W. B., Horne, R. N., and Nordlie, R. C. (1968) Biochemistry 7, 3997-4004). The second order rate constant calculated for formation of glucose 6-vanadate at pH 7.0 is 2.4 M-1 s-1. The corresponding values for glucose 6-phosphate and glucose 6-arsenate formation are approximately 9 X 10(-11) M-1 s-1 and 6.3 X 10(-6) M-1 s-1 (Lagunas, R. (1980) Arch. Biochem. Biophys. 205, 67-75).