PURIFICATION AND CHARACTERIZATION OF AZOTOBACTER-VINELANDII GLUCOSE-6-PHOSPHATE-DEHYDROGENASE - DUAL COENZYME SPECIFICITY

Azotobacter vinelandii glucose-6-phosphate dehydrogenase isolated from cell sonicates was purified 81-fold to electrophoretic homogeneity and a specific activity of 73 units/mg protein using ion-exchange and Matrex Dye chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular exclusion chromatography indicated the enzyme to be a tetramer composed of 52,000 M(r) subunits. The enzyme utilized both NAD and NADP as coenzymes with K-m values of 220 and 50 mu M, respectively. In addition, the purified enzyme functioned well with the thionicotinamide analogs of NAD and NADP. A sigmoidal response was observed in studies of the effect of glucose 6-phosphate concentration on initial velocities. Evidence in support of one enzyme with dual coenzyme specificity was obtained in purification, thermodenaturation, and inhibitor studies. The enzyme exhibited a pH optimum of 8.5. Coenzyme-competitive inhibition was observed with nine adenosine derivatives with no significant selectivity shown for 2'-phosphoryl derivatives. K-i values for product inhibition by NADH and NADPH were higher than the K-m values for the respective oxidized forms of the coenzymes. (C) 1995 Academic Press, Inc.