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Transient-phase kinetic studies on the nucleotide binding to 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 using fluorescence stopped-flow procedures
| Content Provider | Scilit |
|---|---|
| Author | Ueda, Shigeru Oda, Masayuki Imamura, Shigeyuki Ohnishi, Masatake |
| Copyright Year | 2004 |
| Description | Journal: Journal of Biological Inorganic Chemistry 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. |
| Related Links | http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.2004.04089.x/pdf |
| Ending Page | 1780 |
| Page Count | 7 |
| Starting Page | 1774 |
| ISSN | 09498257 |
| e-ISSN | 14321327 |
| DOI | 10.1111/j.1432-1033.2004.04089.x |
| Journal | Journal of Biological Inorganic Chemistry |
| Issue Number | 9 |
| Volume Number | 271 |
| Language | English |
| Publisher | Wiley-Blackwell |
| Publisher Date | 2004-05-01 |
| Access Restriction | Open |
| Subject Keyword | Journal: Journal of Biological Inorganic Chemistry Analytical Chemistry Aldo‐keto Reductase Superfamily Nucleotide‐binding Fluorescence Stopped‐flow Method 3α‐hydroxysteroid Dehydrogenase Short‐chain Dehydrogenase/reductase Superfamily |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry Inorganic Chemistry |
