Knowledge of the partitioning of the putative dienol intermediate (2) by steroid isomerase (KSI) (Hawkinson et al. 1991), in conjunction with various steady-state kinetic parameters, allows elucidation of the detailed free energy profile for the KSI-catalyzed conversion of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (3). This free energy profile shows four kinetically significant energy barriers (substrate binding, the two chemical steps, and dissociation of product) that must be traversed upon conversion of 1 to 3. Thus, no single step of the catalytic cycle is cleanly rate-limiting. The source of the catalytic power of KSI is discussed via comparison of the free energy profile for the KSI-catalyzed isomerization with those for the acetate-catalyzed isomerization and the aqueous reaction at pH 7. Similarities between the energetics of the KSI-catalyzed and triosephosphate isomerase catalyzed reactions are also noted.

The dissociation constant (KD) for the complex of the intermediate dienol (2) and the D38N mutant of 3-oxo-delta 5-steroid isomerase (D38N.2) has been determined for the isomerization of 5-androstene-3,17-dione (1). KD for D38N.2 is pH-dependent, with values of 6 nM at pH 6.9, 51 nM at pH 5.8, and 59 nM at pH 5.2. These values of KD are used to estimate the pH-independent dissociation constant (0.7 +/- 0.3 microM) for the complex of dienol and wild-type (WT) enzyme. The internal equilibrium constant (Kint = 0.3 +/- 0.2) for the interconversion of bound substrate (WT.1) and bound intermediate (WT.2) was then calculated for WT using its KD, the values for the external equilibrium constant for 1<-->2, and the dissociation constant of the enzyme substrate complex (KS). The dissociation constant (KD) for the complex of equilenin (4) with WT, D38E, and D38N enzymes was also determined at pH values from 4 to 7. For the complex of 4 with D38N (D38N.4), KD is pH-dependent with an apparent pKa of about 4.5, whereas KD for both WT.4 and D38E.4 is pH-independent. These values are used to give two additional estimates of the internal equilibrium constant for WT (Kint = 0.5 and 0.01). Analysis of these results in terms of Marcus formalism leads to the conclusion that the primary function of the enzyme is to decrease the thermodynamic barrier to formation of the intermediate by lowering delta Gzero by about 10 kcal/mol. In contrast, the intrinsic free energy of activation (delta G++int) is only decreased by about 3 kcal/mol. These results are discussed in terms of competing theories of enzymatic enolization.

3-Oxo-Delta 5-steroid isomerase (Delta 5-3-ketosteroid isomerase, KSI; EC 5.3.3.1) catalyzes the conversion of a variety of beta, gamma-unsaturated 3-oxosteroids to their corresponding alpha, beta-unsaturated isomers at rates that approach the diffusion limit for specific substrates. The reaction proceeds through a dienolate intermediate, with two amino acid residues (Asp-38 and Tyr-14) known to be involved in catalysis. When the complete three-dimensional structure of KSI was determined recently by NMR methods, an additional polar residue (Asp-99) was found in the active site and this group was shown to be important for catalytic activity. In this work, we examine the properties of several mutant KSIs to determine the nature of catalysis by Asp-99 of KSI. The electrophoretic mobilities of wild-type (WT) KSI and several mutants (D99A, D99N, D38N, and D38N/D99A) on native gels were determined at pH values ranging from 6.0 to 8.5. The results demonstrate that the pKa of Asp-99 is >8.5 in wild-type KSI. The pH-rate profiles for the D99A, D99N, and D38H/D99A mutants of KSI were also determined. For all three mutants, kcat and kcat/KM do not decrease at high pH, in contrast to those for WT and D38H, which lose activity above pH 9 and 8, respectively. Mutation of Asp-99 to Asn decreases kcat for the substrate 5-androstene-3,17-dione by 27-fold and kcat/Km by 23-fold, substantially less than the loss of activity (3000-fold in kcat and 2200-fold in kcat/Km) observed when Asp-99 is mutated to Ala, consistent with a hydrogen bonding role for Asp-99. Taken together, these results provide evidence that Asp-99 participates in catalysis in its protonated form, with a pKa of>9 in WT and approximately 8.5 in the D38H mutant. Asp-99 likely donates a hydrogen bond to O-3 of the steroid, helping to stabilize the transition state(s) of the KSI-catalyzed reaction.

Rate and equilibrium constants for the deprotonation of a series of phenyl-substituted 2-tetralones in aqueous sodium hydroxide have been determined. A Brønsted plot of log k for deprotonation vs pK a of the appropriate 2-tetralone is linear with a slope (−α) of −0.60 ± 0.01, except for the point corresponding to 6-nitro-2-tetralone (1b). The negative deviation of 1b from the correlation indicates that the transition state for deprotonation of 2-tetralone is imbalanced, with delocalization of charge into the phenyl ring lagging behind proton transfer. A semiquantitative assessment of the charge distribution in both the fully formed anion and the transition state for deprotonation was calculated from these results and 13C NMR spectra of the 2-tetralone anion in methanol/water mixtures. Although approximately twice as much negative charge is localized on the oxygen than on the enolate carbon in the anion, slightly more charge is on the enolate carbon in the transition state.