EVALUATION OF THE INTERNAL EQUILIBRIUM-CONSTANT FOR 3-OXO-DELTA(5)-STEROID ISOMERASE USING THE D38E AND D38N MUTANTS - THE ENERGETIC BASIS FOR CATALYSIS

The dissociation constant (K-D) for the complex of the intermediate di enol (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-dio ne (1). K-D 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 K-D are used to estimate the pH-independent dissociation constant (0.7 +/- 0.3 mu M) for the complex of dienol and wild-type (WT) enzyme. The internal equi librium constant (K-int = 0.3 +/- 0.2) for the interconversion of boun d substrate (WT.1) and bound intermediate (WT.2) was then calculated f or WT using its K-D, the values for the external equilibrium constant for 1 reversible arrow 2, and the dissociation constant of the enzyme substrate complex (K-S). The dissociation constant (K-D) for the compl ex of equilenin (4) with WT, D38E, and D38N enzymes was also determine d at pH values from 4 to 7. For the complex of 4 with D38N (D38N.4), K -D is pH-dependent with an apparent pK(a) of about 4.5, whereas K-D fo r both WT.4 and D38E.4 is pH-independent. These values are used to giv e two additional estimates of the internal equilibrium constant for WT (K-int = 0.5 and 0.01). Analysis of these results in terms of Marcus formalism leads to the conclusion that the primary function of the enz yme is to decrease the thermodynamic barrier to formation of the inter mediate by lowering Delta G degrees by about 10 kcal/mol. In contrast, the intrinsic free energy of activation (Delta G(int)(double dagger)) is only decreased by about 3 kcal/mol. These results are discussed in terms of competing theories of enzymatic enolization.