title: Mechanism of proton transfer in the isomerization of 5-androstene-3, 17-dione by 3-oxo-delta 5-steroid isomerase and its D38E mutant.
abstract: The stereochemistry of proton transfer in the isomerization of [4 beta-2H]-5-androstene-3,17- dione (1d) to 4-androstene-3,17-dione (3) catalyzed by 3-oxo-delta 5-steroid isomerase (KSI) has been reinvestigated. In H2O, approximately 65% of the label is retained in the product (3); of this, one-third is at C-4 and two-thirds at C-6 beta. When the same reaction is catalyzed by the D38E mutant of KSI, ca. 60% of the label is retained in the product, but almost all of it is at C-4. These reactions run in deuterium oxide result in 13% incorporation of a second deuterium with the wild type (WT) enzyme and 75% incorporation with the D38E mutant. When unlabeled 1 is isomerized in D2O, there is little incorporation of deuterium with WT (ca. 5 at. %) but substantial incorporation with D38E (130 at. %). These results are consistent with competitive abstraction of both the C-4 alpha and C-4 beta protons, as proposed by Viger et al. [(1981) J. Am. Chem. Soc. 103, 4151], and demonstrate that the KSI reaction is not completely stereospecific. A mechanism is proposed to account for these observations.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, Baltimore 21228-5398, USA.
authors.forename: M E
authors.initials: ME
authors.lastname: Zawrotny
authors.forename: D C
authors.initials: DC
authors.lastname: Hawkinson
authors.forename: G
authors.initials: G
authors.lastname: Blotny
authors.forename: R M
authors.initials: RM
authors.lastname: Pollack
mesh_terms: D000735:Androstenedione / Q000737:chemistry* / Q000378:metabolism*; D003903:Deuterium; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D017354:Point Mutation; D011522:Protons; D013237:Stereoisomerism; D013251:Steroid Isomerases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011522:Protons; D000735:Androstenedione; C003528:5-androstene-3,17-dione; D003903:Deuterium; D013251:Steroid Isomerases; C022660:steroid delta-isomerase