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Báo cáo khoa học: to 3a-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 using fluorescence stopped-flow procedures

The dual nucleotide cofactor-specific enzyme, 3a-hydroxy-steroid dehydrogenase (3a-HSD)from Pseudomonassp. B-0831, is a member of the short-chain dehydrogenase/ reductase (SDR)superfamily. Transient-phase kinetic stud-ies using the fluorescence stopped-flow method were con-ducted with 3a-HSD to characterize the nucleotide binding mechanism. The binding of oxidized nucleotides, NAD + , NADP + and nicotinic acid adenine dinucleotide (NAAD + ), agreed well with a one-step mechanism, while that of reduced nucleotide, NADH, showed a two-step mechanism. . | Eur. J. Biochem. 271 1774-1780 2004 FEBS 2004 doi 10.1111 j.1432-1033.2004.04089.x Transient-phase kinetic studies on the nucleotide binding to 3a-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 using fluorescence stopped-flow procedures Shigeru Ueda1 Masayuki Oda2 Shigeyuki Imamura1 and Masatake Ohnishi2 1 Department Diagnostics Research and Development Division of Fine Chemicals and Diagnostics Asahi Kasei Pharma Corporation Shizuoka Japan 2 Department of Cellular Macromolecule Chemistry Graduate School of Agriculture Kyoto Prefectural University Kyoto Japan The dual nucleotide cofactor-specific enzyme 3a-hydroxy-steroid dehydrogenase 3a-HSD from Pseudomonas sp. B-0831 is a member of the short-chain dehydrogenase reductase SDR superfamily. Transient-phase kineiic ttud-ies using the fluorescence stopped-flow method were conducted with 3a-HSD to characterize the nucleotide binding mechanism. The binding of oxidized nucleotides NAD NADP and nicotinic acid adenine dinucleotide NAAD agreed well with a one-step mechanism while that of reduced nucleotide NADH showed a two-step mechanism. This difference draws attention to previous characteristic findings on rat liver 3a-HSD which is a member of the aldo-keto reductase AKR superlomily. Although functionally similar AKRs are structurally different from SDRs. The dissociation rate constants associated with the enzyme-nucleotide complex formation were larger than the kcat values for either oxidation or reduction of substrates indicating that the release of cofactors is not rate-limiting overall. It should also be noted that kcat for a substrate cholic acid with NADP was only 6 of that with NAD and no catalytic activity was detectable with NAAD despite the similar binding affinities of nucleotides. These results suggest that a certain type of nucleotide can modulate nucleotide-binding mode and further the catalytic function of the enzyme. Keywords aldo-keto reductase superfamily nucleotide-binding fluorescence .