Substituent effects on the binding of phenols to the D38N mutant of 3-oxo-delta5-steroid isomerase. A probe for the nature of hydrogen bonding to the intermediate.
The nature of hydrogen bonding to the intermediate of the reaction catalyzed by 3-oxo-Delta5-steroid isomerase (KSI) was investigated. Substituted phenols bind tightly to the active site of the D38N mutant of KSI, and are analogs of the intermediate dienol. These D38N-phenol complexes exhibit fluorescence, NMR, and UV spectral characteristics similar to D38N complexed with phenolic steroids. The binding of phenols to D38N is satisfactorily described by the modified Bronsted equation: log KD = 0.85(pKa) - 0.63 pi - 6.3 (n = 10, r = 0.967), where KD is the dissociation constant of the complex and pi is the hydrophobicity parameter for the phenol substituent. The high value of the Bronsted alpha (0.85 +/- 0.08) indicates that the negative charge in the D38N-phenol complex, and by implication in the KSI-intermediate complex, is localized almost exclusively on the bound ligand. It is concluded that stabilization of the anionic (dienolate) intermediate is provided by ordinary hydrogen bonds from the enzyme acids Tyr-14 and Asp-99, rather than low-barrier hydrogen bonds.