3-Oxo-Delta(5)-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(double dagger)) of similar to18 kcal/mol. To evaluate whether the decrease in DeltaG(double dagger) 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 k(cat)/K-M versus 1/T gives the activation enthalpy (DeltaH(double dagger) = 5.9 kcal/mol) and activation entropy (TDeltaS(double dagger) = -2.6 kcal/mol). Relative to catalysis by acetate, D38E reduces DeltaH(double dagger) by similar to10 kcal/mol and increases TDeltaS(double dagger) by similar to5 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.