Studies of the Mechanism of the Δ⁵-3-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, k_cat(H)/k_cat(4β-D) is 6.1 in H₂O and 6.3 in D₂O. The solvent isotope effect, k_cat(H₂O)/k_cat(D₂O), is 1.6 for both the 4β-H and 4β-D substrates. Mutation of Tyr-55 to Phe lowers k_cat 4.3-fold; k_cat(H)/k_cat(4β-D) is 5.3 in H₂O and 5.9 in D₂O, and k_cat(H₂O)/k_cat(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 K_m 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 k_cat/K_m 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 k_cat/K_m 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 k_cat of mutation of these catalytic residues [Kuliopulos, A., Talalay, P., & Mildvan, A. S. (1990) Biophys. J. 57, 39a].