Binding of 2-naphthols to D38E mutants of 3-oxo-Delta(5)-steroid isomerase: Variation of ligand ionization state with the nature of the electrophiliccomponent
Ip. Petrounia et al., Binding of 2-naphthols to D38E mutants of 3-oxo-Delta(5)-steroid isomerase: Variation of ligand ionization state with the nature of the electrophiliccomponent, BIOCHEM, 39(1), 2000, pp. 110-116
3-Oxo-Delta(5)-steroid isomerase (KSI) catalyzes the isomerization of a var
iety of 3-oxo-Delta(5)-steroids to their conjugated Delta(4) isomers. The m
echanism involves sequential enolization and ketonization, with Asp-38 acti
ng to transfer a proton from C-4 to C-6 through a dienol(ate) intermediate.
We have previously proposed that this intermediate is anionic, with stabil
ization provided from direct hydrogen bonding from Tyr-14 and Asp-99 to the
oxygen of the steroid. In this work, we analyze the binding of substituted
2-naphthols, which are analogues of the intermediate dienol, to the D38E K
SI mutant and the corresponding double mutants lacking one of the two elect
rophilic groups (D38E/Y14F and D38E/D99A). The binding of these naphthols t
o the mutant KSIs at pH 7 is described by the modified Bronsted equation: l
og K-D = alpha(pK(a)) + constant, where K-D is the dissociation constant of
the complex. The high value of alpha for D38E (alpha = 0.87 +/- 0.06) indi
cates that the negative charge in these D38E-naphthol complexes is localize
d almost exclusively on the bound ligand. In contrast, values of a fur the
double mutants (alpha = 0.28 +/- 0.02 for D38E/Y14F and alpha = 0.25 +/- 0.
02 for D38E/D99A) are consistent with very little negative charge on the ox
ygen of the bound naphthol. Ultraviolet spectra of 5-nitro-2-naphthol and t
he fluorescence spectra of equilenin bound to these mutants support this in
terpretation. Extrapolation of these results to the intermediate in the cat
alytic reaction suggests that for the reaction with D38E, the intermediate
is a negatively charged dienolate with hydrogen bonding from both Tyr-14 an
d Asp-99. Removal of either one of these H-bond donors (Tyr-14 or Asp-99) c
auses destabilization of the anion and results in a dienol enzyme-intermedi
ate complex rather than a dienolate.