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 Km values of 220 and 50 microM, 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. Ki values for product inhibition by NADH and NADPH were higher than the Km values for the respective oxidized forms of the coenzymes.
Anderson
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Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308, USA.
Anderson
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D000227:Adenine Nucleotides / Q000494:pharmacology; D016948:Azotobacter vinelandii / Q000201:enzymology*; D001667:Binding, Competitive; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism*; D009249:NADP / Q000031:analogs & derivatives / Q000378:metabolism*; D013379:Substrate Specificity
D000227:Adenine Nucleotides; D046911:Macromolecular Substances; D009243:NAD; C015541:thionicotinamide adenine dinucleotide phosphate; C003451:thionicotinamide adenine dinucleotide; D009249:NADP; D005954:Glucosephosphate Dehydrogenase