STUDIES OF THE MECHANISM OF THE DELTA-5-3-KETOSTEROID ISOMERASE REACTION BY SUBSTRATE, SOLVENT, AND COMBINED KINETIC DEUTERIUM-ISOTOPE EFFECTS ON WILD-TYPE AND MUTANT ENZYMES

Δ5-3-Ketosteroid isomerase (EC 5.3.3.1) catalyzes the isomerization of Δ5-3-ketosteroids to Δ4-3-ketosteroids by a conservative tautomeric transfer of the 4β-proton to the 6β-position using Tyr-14 as a general acid and Asp-38 as a general base [Kuliopulos, A., Mildvan, A. S., Shortle, D., & Talalay, P. (1989) Biochemistry 28, 149]. On deuteration of the 4β-position (97.0%) of the substrate, kcat(H)/kcat(4β-D) is 6.1 in H2O and 6.3 in D2O. The solvent isotope effect, kcat(H2O)/kcat(D2O), is 1.6 for both the 4β-H and 4β-D substrates. Mutation of Tyr-55 to Phe lowers kcat 4.3-fold; kcat(H)/kcat(4β-D) is 5.3 in H2O and 5.9 in D2O, and kcat(H2O)/kcat(D2O) with the 4β-H and 4β-D substrates is 1.5 and 1.7, respectively, indicating concerted general acid-base catalysis in either the enolization or the ketonization step of both the wild-type and the Tyr-55 → Phe (Y55F) mutant enzymes. An additional slow step occurs with the Y55F mutant. Smaller isotope effects on Km are used to estimate individual rate constants in the kinetic schemes of both enzymes. On deuteration of the 4α-position (88.6%) of the substrate, the secondary isotope effect on kcat/Km corrected for composition is 1.11 ± 0.02 with the wild-type enzyme and 1.12 ± 0.02 with the Y55F mutant. These effects decrease to 1.06 ± 0.01 and 1.07 ± 0.01, respectively, when the 4β-position is also deuterated, thereby establishing these to be kinetic (rather than equilibrium) secondary isotope effects and to involve a proton-tunneling contribution. Deuteration of the 6-position of the substrate (92.0%) produces no kinetic isotope effects on kcat/Km with either the wild-type (1.00 ± 0.01) or the Y55F mutant (1.01 ± 0.01) enzyme. Since a change in hybridization from sp3 to sp2 occurs at C-4 only during enolization of the substrate and a change in hybridization at C-6 from sp2 to sp3 occurs only during reketonization of the dienol intermediate, enolization of the substrate constitutes the concerted rate-limiting step. Concerted enolization is consistent with the right angle or antarafacial orientations of Tyr-14 and Asp-38 with respect to the enzyme-bound substrate and with the additive effects on kcat of mutation of these catalytic residues [Kuliopulos, A., Talalay, P., & Mildvan, A. S. (1990) Biophys. J. 57, 39a]. © 1990, American Chemical Society. All rights reserved.