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A Complete Kinetic Mechanism of Rat 3α‐Hydroxysteroid Dehydrogenase
Author(s) -
Cooper William C.,
Jin Yi,
Penning Trevor M.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a480
Subject(s) - steroid , chemistry , reaction rate constant , substrate (aquarium) , stereochemistry , steroid hormone , steady state (chemistry) , ternary complex , dehydrogenase , receptor , kinetics , enzyme , hormone , biochemistry , biology , physics , quantum mechanics , ecology
Hydroxysteroid dehydrogenases (HSDs) convert potent steroid hormones into their inactive metabolites and regulate ligand occupancy of steroid hormone receptors. Rat 3α‐HSD is the most thoroughly characterized in the aldo‐keto reductase (AKR) superfamily and is a model for studying these reactions. We have determined eighteen microscopic rate constants for an ordered bi‐bi mechanism in which androstane‐3, 17‐dione is reversibly converted to androsterone using both steady‐state and transient state kinetic approaches. Rapid mixing of NADP(H) with the enzyme indicated the formation of a tight bound complex having an off rate that approached k cat . However upon examining rate constants for substrate binding the off rate for steroid from the ternary complex formed was ruled not rate‐determining. In multiple turnover experiments for steroid oxidation a large burst was noted and consistent with product release being rate determining. In multiple turnover experiments for steroid reduction traces were characterized by a small but discernable burst which was accompanied by a primary KIE. By contrast no KIE was observed in the steady‐state. Thus the chemical step was not rate determining for steroid reduction. For the substrate pair studied, the chemical rates in the reduction direction were slow, but the release of products was slower. Prior studies using an alternate substrate pair DHT/3α‐diol showed that chemistry in the reduction direction was rate‐determining. Therefore, in reactions which regulate steroid hormone action the rate‐determining step is dependent upon the substrate pair. This work was supported by NIH Grant DK47015 to TMP.