Kinetic studies of Haemophilus influenzae 6-phosphogluconate dehydrogenase.
Haemophilus influenzae 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ 2-oxidoreductase (decarboxylating), EC 1.1.1.44) was purified 308-fold to electrophoretic homogeneity with a 16% recovery through a five-step procedure involving salt fractionation and hydrophobic and affinity chromatography. The purified enzyme was demonstrated to be a dimer of Mr 70,000, and to catalyze a sequential reaction process. The enzyme was NADP-specific and kinetic parameters for the oxidation of 6-phosphogluconate were determined for NADP and four structural analogs of NADP. Coenzyme-competitive inhibition by adenosine derivatives was significantly enhanced by the presence of a 2'-phosphoryl group consistent with the observed coenzyme specificity of the enzyme. The purified enzyme was effectively inhibited by 3-aminopyridine adenine dinucleotide phosphate, but at concentrations higher than that observed to inhibit growth of the organism. Rates of inactivation of the enzyme by N-ethylmaleimide were suggestive of sulfhydryl involvement in the reaction catalyzed.
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University Blacksburg 24061.
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D000227:Adenine Nucleotides / Q000494:pharmacology; D001667:Binding, Competitive; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D005942:Gluconates / Q000378:metabolism; D006193:Haemophilus influenzae / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009243:NAD / Q000031:analogs & derivatives / Q000494:pharmacology; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D010734:Phosphogluconate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011489:Protein Denaturation
D000227:Adenine Nucleotides; D005942:Gluconates; D046911:Macromolecular Substances; D009243:NAD; C002098:3-aminopyridine adenine dinucleotide; D009249:NADP; D010734:Phosphogluconate Dehydrogenase; D005033:Ethylmaleimide; C008884:6-phosphogluconic acid