MECHANISM OF PROTON-TRANSFER IN THE ISOMERIZATION OF 5-ANDROSTENE-3,17-DIONE BY 3-OXO-DELTA(5)-STEROID ISOMERASE AND ITS D38E MUTANT

The stereochemistry of proton transfer in the isomerization of [4 beta -H-2]-5-androstene-3,17-dione (1d) to 4-androstene-3,17-dione (3) cata lyzed by 3-oxo-Delta(5)-steroid isomerase (KSI) has been reinvestigate d. 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 l abel 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 sec ond deuterium with the wild type (WT) enzyme and 75% incorporation wit h the D38E mutant. When unlabeled 1 is isomerized in D2O, there is lit tle incorporation of deuterium with WT (ca. 5 at. %) but substantial i ncorporation 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. Sec. 103, 4151], and de monstrate that the KSI reaction is not completely stereospecific. A me chanism is proposed to account for these observations.