Kinetic studies of Haemophilus influenzae 6-phosphogluconate dehydrogenase |
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| Affiliation: | 1. Department of Orthopaedic Surgery, The First Affiliated Hospital of Nanjing Medical University, China;2. Department of Biostatistics, School of Public Health, Nanjing Medical University, Nanjing 210029, Jiangsu, China;3. Department of Orthopaedic Surgery, Yangzhou Hongquan Hospital, Yangzhou 225243, Jiangsu, China;1. Department of Biochemistry and Molecular Medicine, University of California at Davis, School of Medicine, Sacramento, CA 95817, USA;2. Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children-Northern California, UC Davis School of Medicine, Sacramento, CA 95817, USA;1. Centre for Cancer Prevention, Wolfson Institute of Preventive Medicine, Queen Mary University of London, London, UK;1. Gene Center, Department of Biochemistry and Center for Integrated Protein Science Munich (CiPSM), Feodor-Lynenstr. 25, 81377 München, Germany;2. Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany;1. INSA Centre Val de Loire, Université François Rabelais de Tours, LMR EA 2640, Campus de Blois, 3 Rue de la Chocolaterie, CS 23410, 41034 Blois Cedex, France;2. LMA, CNRS, UPR 7051, Centrale Marseille, Aix-Marseille Univ, F-13420 Marseille Cedex 20, France |
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| Abstract: | Haemophilus influenzae 6-phosphogluconate dehydrogenase (6-phospho-d-gluconate:NADP+ 2-oxidoreductase (decar☐ylating), 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. |
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