SUBSTITUENT EFFECTS ON THE BINDING OF PHENOLS TO THE D38N MUTANT OF 3-OXO-DELTA(5)-STEROID ISOMERASE - A PROBE FOR THE NATURE OF HYDROGEN-BONDING TO THE INTERMEDIATE

The nature of hydrogen bonding to the intermediate of the reaction cat alyzed by 3-oxo-Delta(5)- steroid isomerase (KSI) was investigated. Su bstituted phenols bind tightly to the active site of the D38N mutant o f KSI, and are analogs of the intermediate dienol. These D38N-phenol c omplexes exhibit fluorescence, NMR, and UV spectral characteristics si milar to D38N complexed with phenolic steroids. The binding of phenols to D38N is satisfactorily described by the modified Bronsted equation : log K-D = 0.85(pK(a)) -0.63 pi -6.3 (n = 10, r = 0.967), where K-D i s the dissociation constant of the complex and pi is the hydrophobicit y 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 tha t stabilization of the anionic (dienolate) intermediate is provided by ordinary hydrogen bonds from the enzyme acids Tyr-14 and Asp-99, rath er than low-barrier hydrogen bonds.