Temperature Effects on the Catalytic Activity of the D38E Mutant of 3-Oxo-Δ5-Steroid Isomerase: Favorable Enthalpies and Entropies of Activation Relative to the Nonenzymatic Reaction Catalyzed by Acetate Ion

Abstract

3-oxo-Delta5-steroid isomerase (ketosteroid isomerase, KSI) catalyzes the isomerization of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (3) via a dienolate intermediate (2-). KSI catalyzes this conversion about 13 orders of magnitude faster than the corresponding reaction catalyzed by acetate ion, a difference in activation energy (DeltaG) of approximately 18 kcal/mol. To evaluate whether the decrease in DeltaG by KSI is due to enthalpic or entropic effects, the activation parameters for the isomerization of 1 catalyzed by the D38E mutant of KSI were determined. A linear Arrhenius plot of kcat/KM versus 1/T gives the activation enthalpy (DeltaH = 5.9 kcal/mol) and activation entropy (TDeltaS = -2.6 kcal/mol). Relative to catalysis by acetate, D38E reduces DeltaH by approximately 10 kcal/mol and increases TDeltaS by approximately 5 kcal/mol. The activation parameters for the microscopic rate constants for D38E catalysis were also determined and compared to those for the acetate ion-catalyzed reaction. Enthalpic stabilization of 2- and favorable entropic effects in both chemical transition states by D38E result in an overall energetically more favorable enzymatic reaction relative to that catalyzed by acetate ion.