Kinetic competence of an externally generated dienol intermediate with steroid isomerase

The putative intermediate dienol (2) in the steroid isomerase (KSI) catalyzed conversion of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (3) has been independently generated and tested as a substrate for KSI. At pH 7, dienol 2 is converted by KSI to a mixture of 1 (46%) and 3 (54%). The apparent second-order rate constant for reaction of 2 with KSI to produce 3 (kappa cat/Km = 2.3 x 10(8) M-1 s-1) is similar to that for reaction of 1 with KSI (kappa cat/Km = 2.1 x 10(8) M-1 s-1), demonstrating that 2 is kinetically competent. Isomerization of 1 by KSI in D2O gives only 5% of solvent deuterium incorporated into the product 3. When 2 reacts with KSI in D2O, and the product 3 is isolated (from direct reaction of 2 and from subsequent conversion of the 1 initially formed), ca. 80 atom % deuterium is located at C-6 beta, confirming that protonation of the dienol by KSI occurs at the same face as the proton transfer in the KSI catalyzed reaction of 1 to 3.