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.
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