Open AccessTransient‐phase kinetic studies on the nucleotide binding to 3α‐hydroxysteroid dehydrogenase from Pseudomonas sp. B‐0831 using fluorescence stopped‐flow procedures
Author(s) -
Ueda Shigeru,
Oda Masayuki,
Imamura Shigeyuki,
Ohnishi Masatake
Publication year - 2004
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.2004.04089.x
Subject(s) - nucleotide , nad+ kinase , cofactor , dehydrogenase , biochemistry , enzyme , stereochemistry , chemistry , reductase , hydroxysteroid dehydrogenase , binding site , biology , gene
The dual nucleotide cofactor‐specific enzyme, 3α‐hydroxysteroid dehydrogenase (3α‐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 3α‐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 3α‐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.