Transient-phase kinetic studies on the nucleotide binding to 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 using fluorescence stopped-flow procedures.
The dual nucleotide cofactor-specific enzyme, 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) from Pseudomonas sp. B-0831, is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Transient-phase kinetic studies using the fluorescence stopped-flow method were conducted with 3alpha-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 3alpha-HSD, which is a member of the aldo-keto reductase (AKR) superfamily. Although functionally similar, AKRs are structurally different from SDRs. The dissociation rate constants associated with the enzyme-nucleotide complex formation were larger than the k(cat) 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 k(cat) 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.
Ueda
Shigeru
S
Department Diagnostics Research and Development, Division of Fine Chemicals and Diagnostics, Asahi Kasei Pharma Corporation, Shizuoka, Japan.
Oda
Masayuki
M
Imamura
Shigeyuki
S
Ohnishi
Masatake
M
D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific) / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
D009243:NAD; D009249:NADP; C018348:nicotinic acid adenine dinucleotide; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)