REACTION ENERGETICS OF A MUTANT 3-OXO-DELTA(5)-STEROID ISOMERASE WITH AN ALTERED ACTIVE-SITE BASE (D38E)

3-Oxo-Delta(5)-steroid isomerase (KSI) catalyzes the isomerization of a variety of 3-oxo-Delta(5)-steroids to their conjugated Delta(4)- isomers through the formation of an intermediate dienol. Mutation of the catalytic base (Asp-38) to Glu (D38E) has been found to reduce k(cat)/K-m for the isomerization of 5-androstene-3, 17-dione (1) to 4-androstene-3,17-dione (3) by about 300-fold (Zawrotny et al., 1991). The free energy profile for the D38E enzyme was determined from a combination of steady state kinetics and stopped-flow kinetics with the independently generated dienol intermediate (2). A comparison of the energetics of D38E with that of the wild type enzyme (WT) shows that the only significant difference is a reduction in the rates of the chemical steps for the interconversion of 1, 2, and 3 on the enzyme surface by about 10(3)-fold for D38E. The relative energy levels for all bound species are nearly identical for WT and D38E, whereas the transition states for both enolization and ketonization are destabilized by 3-4 kcal/mol. The effect of the D38E mutation on the energetics of KSI is comparable to the corresponding effect of the E165D mutation on the energetics of triosephosphate isomerase (TIM).