Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole

Abstract

The catalytic importance of enzyme active-site interactions is frequently assessed by mutating specific residues and measuring the resulting rate reductions. This approach has been used in bacterial ketosteroid isomerase to probe the energetic importance of active-site hydrogen bonds donated to the dienolate reaction intermediate. The conservative Tyr16Phe mutation impairs catalysis by 10(5)-fold, far larger than the effects of hydrogen bond mutations in other enzymes. However, the less-conservative Tyr16Ser mutation, which also perturbs the Tyr16 hydrogen bond, results in a less-severe 10(2)-fold rate reduction. To understand the paradoxical effects of these mutations and clarify the energetic importance of the Tyr16 hydrogen bond, we have determined the 1.6-A resolution x-ray structure of the intermediate analogue, equilenin, bound to the Tyr16Ser mutant and measured the rate effects of mutating Tyr16 to Ser, Thr, Ala, and Gly. The nearly identical 200-fold rate reductions of these mutations, together with the 6.4-A distance observed between the Ser16 hydroxyl and equilenin oxygens in the x-ray structure, strongly suggest that the more moderate rate effect of this mutant is not due to maintenance of a hydrogen bond from Ser at position 16. These results, additional spectroscopic observations, and prior structural studies suggest that the Tyr16Phe mutation results in unfavorable interactions with the dienolate intermediate beyond loss of a hydrogen bond, thereby exaggerating the apparent energetic benefit of the Tyr16 hydrogen bond relative to the solution reaction. These results underscore the complex energetics of hydrogen bonding interactions and site-directed mutagenesis experiments.

Fig. 1.

KSI substrates and reaction intermediate analogue. (A) Reaction mechanism for isomerization of 5(10)-estrene-3,17-dione (5(10)-EST). (B) Isomerization of 5-androstene-3,17-dione (5-AND). (C) Schematic depiction of equilenin bound at the KSI active site.

Fig. 2.

Effects of Tyr16 mutations on KSI activity (k_cat). Values and errors are averages and standard deviations from three or more independent measurements at different enzyme concentrations and are from Table S2. The indicated values for the Tyr16Ser and Tyr16Phe mutants are identical within error to values we recently reported in a separate publication (16).

Fig. 3.

Structural studies of KSI. (A) Sigma-A-weighted 2F_o - F_c electron density map (contoured at 1.5σ) from the 1.6-Å resolution structure of equilenin bound to Tyr16Ser/Asp40Asn pKSI. Distances are average values (standard deviation ± 0.1 Å) from the four independently refined monomers contained in the asymmetric unit. (B) Superposition of the pKSI Tyr16Ser/Asp40Asn·equilenin structure determined herein (carbon atoms colored cyan), the 1.8-Å resolution structure of unliganded pKSI Tyr16Phe (PDB entry 1EA2, carbon atoms colored violet), and the 1.1-Å resolution structure of equilenin bound to wild-type pKSI (PDB entry 1OH0, carbon atoms colored green). (C) Triangle-shaped sigma-A-weighted 2F_o - F_c electron density (cyan mesh, contoured at 1.5σ) assigned to partially disordered water within the approximately 100 Å³ cavity resulting from the Tyr16Ser mutation. Yellow dashes connect groups within hydrogen-bonding distance of visible electron density to the region of greatest electron density (visible up to 3.3σ). (D) Downfield region of ¹H NMR spectrum of equilenin bound to tKSI Tyr16Phe/Asp40Asn. The peak at approximately 13 ppm is present in spectra of unliganded KSI (11, 25).

Fig. 4.

Binding of 2-fluorophenolate reaction intermediate analogues to pKSI. (A) Structural model of 2-fluorophenolate (carbon atoms in yellow, fluorine in black) bound to pKSI Tyr16Ser (carbon atoms and electron density of disordered water in Ser16 cavity colored cyan) and pKSI Tyr16Phe (carbon atoms colored violet, Phe16 shown as space-filling) generated by superposition of pKSI Asp40Asn·2 - fluorophenolate (PDB entry 3CPO), pKSI Tyr16Ser/Asp40Asn·equilenin (PDB entry 3IPT), and pKSI Tyr16Phe (PDB entry 1EA2). (B) ¹⁹F NMR spectra of 2-fluoro-4-nitrophenolate (pK_a = 6.0) in aqueous solution (pH 12), in the aprotic and low polarity organic solvent THF containing excess triethylamine to form the phenolate anion, and bound to pKSI Tyr16Ser/Asp40Asn or pKSI Tyr16Phe/Asp40Asn.

Fig. 5.

Schematic models for the effects of Tyr16 mutations on KSI catalysis. Shorter hydrogen bonds are depicted with thicker dots. (A) Solution nonenzymatic reaction with water molecules shown as colored dipoles that reorient to solvate localized charge in the dienolate-like transition state. (B) The wild-type oxyanion hole has two positioned enzyme groups that donate hydrogen bonds to the reacting substrate that may strengthen in the transition state. (C) The Tyr16Phe mutation ablates one of the oxyanion hole hydrogen bonds but does not permit water entry, resulting in a hydrophobic surface that desolvates the localized negative charge in the dienolate-like transition state. (D) The Tyr16Ser mutation replaces Tyr16 with a water-filled cavity that provides aqueous-like solvation of the reacting substrate.