Nicotinamide Adenine Dinucleotide- and Nicotinamide Adenine Dinucleotide Phosphate-specific Glucose 6-Phosphate Dehydrogenases of Acetobacter xylinum and Their Role in the Regulation of the Pentose Cycle

Abstract Extracts of Acetobacter xylinum cells grown on glucose or succinate, oxidized glucose 6-phosphate to 6-phosphogluconate in the presence of either NAD or NADP. NAD-linked glucose 6-phosphate dehydrogenase and the corresponding NADP enzyme were completely separated and partially purified by DEAE-cellulose chromatography. The NAD enzyme was optimally active at pH 5.6, and the NADP enzyme at pH 8.2. Each enzyme exhibited strict specificity for its respective coenzyme. The interaction of glucose 6-phosphate and the corresponding coenzyme was that of a typical two-substrate system. The reaction rates with both enzymes were hyperbolic functions of coenzyme and substrate concentrations. The Km values for glucose 6-phosphate and the corresponding coenzyme were 2.5 mm and 0.08 mm for the NAD enzyme and 1.64 mm and 0.04 mm for the NADP enzyme. Magnesium ions did not affect the activity of the enzymes. The NAD enzyme was competitively inhibited by NADH with respect to NAD (Ki = 0.035 mm) and was not affected by NADPH. The NADP enzyme was competitively inhibited by NADPH with respect to NADP (Ki = 0.028 mm) and was insensitive to NADH. ATP inhibited the activity of the NAD glucose 6-phosphate dehydrogenase. The inhibition, which followed classical kinetic patterns, was competitive with respect to glucose 6-phosphate (Ki) = 1.3 mm) and noncompetitive with respect to NAD. The NADP-linked enzyme was insensitive to ATP. It is suggested that the NAD and NADP glucose 6-phosphate dehydrogenases have distinctly different functional roles. The possible relationship between the properties of the two enzymes and regulatory mechanisms in the control of hexose metabolism in A. xylinum is discussed.