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
Ip. Petrounia et Rm. Pollack, 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, Biochemistry, 37(2), 1998, pp. 700-705
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.