Abstract
6-Phospho-D-gluconate dehydrogenase (EC 1.1.1.44) was purified and crystallized from a cell free extract of Gluconobacter suboxydans IFO 12528. Affinity chromatography on a blue-dextran Sepharose 4B column was effective through the purification steps, and a 280-fold purification was achieved with a 56% yield. The crystalline enzyme was homogeneous, as judged by polyacrylamide gel electrophoresis and analytical ultracentrifugation. Although the enzyme from G. suboxydans catalyzed the oxidative decarboxylation of 6-phospho-D-gluconate to D-ribulose-5-phosphate, it differed from enzymes derived from other sources in respect to substrate specificity and molecular properties. Unlike the others, this Gluconobacter enzyme has a requirement for similar amounts of NAD and NADP as coenzymes and the pH optimum of this protein with two coenzymes was samely 6.5, while other enzyme preparations have the optimum pH at 8.0. The molecular weight of this enzyme was 175, 000, and the enzyme was composed of four identical subunits of molecular weight 43, 000. The enzyme activity was inhibited competitively by ATP, D-fructose-1, 6-diphosphate and NAD(P)H. It was shown that the NADPH formed by the action of the enzyme was reoxidized by the old yellow enzyme of the organism.
