Hydrogen Bonding at the Active Site of Δ5-3-Ketosteroid Isomerase

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Abstract
The solution secondary structure of the highly active Y55F/Y88F “Tyr-14-only” mutant of Δ5-3-ketosteroid isomerase complexed with 19-nortestosterone hemisuccinate has been shown to consist of three helices, a six-stranded mixed β-sheet, and five turns. The steroid binds near the general acid, Tyr-14, on helix 1, near the general base, Asp-38, on the first strand of the β-sheet, and on the hydrophobic face of the β-sheet [Zhao, Q., Abeygunawardana, C., & Mildvan, A. S. (1997) Biochemistry 36, 3458−3472]. On this hydrophobic face, Asp-99 is the only polar residue. Free isomerase shows a deshielded exchangeable proton resonance at 13.1 ppm assigned to the NεH of neutral His-100. Its fractionation factor (φ = 0.79) and slow exchange with solvent suggest it to be buried or involved in an H-bond. The binding of dihydroequilenin or estradiol to isomerase induces the appearance of two additional deshielded proton resonances, one at 18.2 ppm assigned to the γ-carboxyl proton of Asp-99, and the other, at 11.6 ppm, assigned to the ζ-OH proton of Tyr-14. While mutation of Asp-99 to Ala results in the disappearance of only the resonance near 18 ppm [Wu, R. W., Ebrahemian, S., Zawrotny, M. E., Thornberg, L. D., Perez-Alverado, G. C., Brothers, P., Pollack, R. M., & Summers, M. F. (1997) Science 276, 415−418], both of these resonances disappear in mutants lacking Tyr-14, suggesting an H-bonded catalytic diad, Asp-99-COOH---Tyr14-OH---O-steroid enolate. The catalytic diad is further supported by NOEs from the β1 and β2 protons of Asp-99 to the ε protons of Tyr-14, and from the ζ-OH proton of Tyr-14 to the γ-carboxyl proton of Asp-99, indicating close proximity of these two residues, and by other data from the literature. A strong, low-barrier H-bond between Asp-99 and Tyr-14 is indicated by the 6.2 ppm deshielding, low fractionation factor (φ = 0.34) and slow exchange of the resonance at 18.2 ppm. A normal H-bond between Tyr-14 and the steroid is indicated by the 1.8 ppm deshielding, fractionation factor of 0.97 and the slow exchange of the resonance at 11.6 ppm. It is suggested that the 104.7-fold contribution of Tyr-14 to catalysis is made possible by strong H-bonding from Asp-99 in the catalytic diad which strengthens general acid catalysis by Tyr-14. It is also noted that highly deshielded proton resonances on enzymes between 15 and 20 ppm, assigned to low-barrier H-bonds, generally involve carboxyl groups.