Kinetics of the Salt Modifications of Glucose 6-Phosphate Dehydrogenase Purified from a Marine Vibrio alginolyticus

Abstract

Glucose 6-phosphate dehydrogenase was partially purified from a marine Vibrio alginolyticus. The enzyme specifically required NADP⁺ as a coenzyme and showed no significant activity toward glucose. Its molecular weight was approximately 100,000 and it consisted of two subunits. The optimum activity was obtained at pH 9.0 and at an ionic strength of 0.1. Polyvalent anions such as sulfate, phosphate, and bicarbonate also stimulated the activity as a result of the increase in ionic strength. High concentrations of salts, and particularly chaotropic anions, inhibited the activity and the order of inhibitory effect followed the chaotropic sequence: SCN−>NO₃⁻>Br⁻>Cl⁻. The inhibition by chaotropic anions was not due to dissociation of the active form of the enzyme to its inactive subunits. The kinetic mechanism of this enzyme followed a sequential ordered mechanism with NADP⁺ combining with the free enzyme first and NADPH being the last product to be released. From kinetic analyses, it was found that the activation of the enzyme on increasing the ionic strength is due to a decrease in the K_m for G-6-P, whereas the inhibitory effect of high concentrations of salts or chaotropic anions is due to an increase in the dissociation constant of NADP⁺-enzyme complex (K_1a). The effect of NO₃⁻ in increasing the K_1a value was found to be due to a decrease in the rate constant for the formation of NADP⁺-enzyme complex, without significantly affecting the rate constant for its dissociation.