NATURE OF THE INTERMEDIATE IN THE 3-OXO-DELTA-5-STEROID ISOMERASE REACTION

The role of Tyr-14 of 3-oxo-DELTA-5-steroid isomerase (KSI) was probed by analysis of the spectra of 3-amino-1,3,5(10)-estratrien-17-beta-ol (4) and equilenin (5) bound to the active site of KSI. The ultraviolet spectrum of 4 bound to KSI is identical to that for 4 in neutral solution. This observation indicates that Tyr-14 does not protonate the amine group of 4 at the active site. By analogy, it is argued that the 3-oxo group of steroid substrates for KSI is not protonated during the reaction. In contrast, the fluorescence excitation spectra of 5 bound to KSI show characteristics of an ionized phenol, even at pH values as low as 3.8. It is concluded that the pK(a) of equilenin is perturbed from its value in solution of 9 to less-than-or-equal-to 3.5 at the active site of KSI. Similarly, the pK(a) of the intermediate dienol in the KSI reaction should be lowered to less-than-or-equal-to 4.5 when it is bound to KSI. Thus, the function of Tyr-14 as an electrophilic catalyst is likely the stabilization of the anion of the dienol by hydrogen bonding rather than by proton transfer.