Δ5-3-Ketosteroid isomerase (EC 220.127.116.11) catalyzes the isomerization of Δ5-3-ketosteroids to Δ4-3-ketosteroids by a conservative tautomeric transfer of the 4β-proton to the 6β-position with 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, 149-159]. Primary, secondary, and combined deuterium kinetic isotope effects establish concerted substrate enolization to be the rate-limiting step with the wild-type enzyme [Xue, L., Talalay, P., & Mildvan, A. S. (1990) Biochemistry 29, 7491-7500]. The product of the fractional kcat values resulting from the Y14F mutation (10−4.7) and the D38N mutation (10−5.6) is comparable (10−10.3) to that of the double mutant Y14F + D38N (≤10−10.4) which is completely inactive. Hence, the combined effects are either additive or synergistic. Quantitatively, similar effects of the two mutations on kcat/KM are found in the double mutant. Despite its inactivity, the Y14F + D38N double mutant forms crystals indistinguishable in form from those of the wild-type enzyme, tightly binds steroid substrates and substrate analogues, and immobilizes a spin-labeled steroid in an orientation indistinguishable from that found in the wild-type enzyme, indicating that the double mutant is otherwise largely intact. It is concluded that the total enzymatic activity of ketosteroid isomerase probably results from the independent and concerted functioning of Tyr-14 and Asp-38 in the rate-limiting enolization step, in accord with the perpendicular or antarafacial orientation of these two residues with respect to the enzyme-bound substrate. Synergistic effects of mutating two residues on kcat and on kcat/KM of enzyme-catalyzed multistep reactions are shown, theoretically, to occur when both residues act independently in the same step, and simple additivity occurs when this step is rate-limiting. Other conditions for additivity of the effects of mutations of kcat and kcat/KM are theoretically explored.
ASJC Scopus subject areas