Purification and Characterization of Azotobacter vinelandii Glucose-6-Phosphate Dehydrogenase: Dual Coenzyme Specificity

• Published in: Archives of biochemistry and biophysics 1995-08, Vol.321 (1), p.94-100
• Main Authors: Anderson, B.M., Anderson, C.D.
• Format: Article
• Language: English
• Subjects: Adenine Nucleotides - pharmacology; AZOTOBACTER VINELANDII; Azotobacter vinelandii - enzymology; Binding, Competitive; Chromatography, Gel; COENZIMAS; COENZYME; Electrophoresis, Polyacrylamide Gel; GLUCOSA 6 FOSFATO DEHIDROGENASA; GLUCOSE 6 PHOSPHATE DESHYDROGENASE; Glucosephosphate Dehydrogenase - chemistry; Glucosephosphate Dehydrogenase - isolation & purification; Glucosephosphate Dehydrogenase - metabolism; INHIBICION; INHIBITION; Kinetics; Macromolecular Substances; Molecular Weight; NAD - analogs & derivatives; NAD - metabolism; NADP - analogs & derivatives; NADP - metabolism; NUCLEOTIDE; NUCLEOTIDOS; PURIFICACION; PURIFICATION; Substrate Specificity
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1995.1372

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 μM, 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.