HYDROGEN-BONDING AT THE ACTIVE-SITE OF DELTA(5)-3-KETOSTEROID ISOMERASE

The solution secondary structure of the highly active Y55F/Y88F ''Tyr- 14-only'' mutant of Delta(5)-3-ketosteroid isomerase complexed with 19 -nortestosterone hemisuccinate has been shown to consist of three heli ces, a six-stranded mixed beta-sheet, and five turns. The steroid bind s near the general acid, Tyr-14, on helix 1, near the general base, As p-38, on the first strand of the beta-sheet, and on the hydrophobic fa ce of the beta-sheet [Zhao, Q., Abeygunawardana, C., & Mildvan, A. S. (1997) Biochemistry 36, 3458-3472]. On this hydrophobic face, Asp-99 i s the only polar residue. Free isomerase shows a deshielded exchangeab le proton resonance at 13.1 ppm assigned to the N epsilon H of neutral His-100. Its fractionation factor (phi = 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 assign ed to the gamma-carboxyl proton of Asp-99, and the other, at 11.6 ppm, assigned to the zeta-OH proton of Tyr-14. While mutation of Asp-99 to Ala results in the disappearance of only the resonance near 18 ppm [W u, R. W., Ebrahemian, S., Zawrotny, M. E., Thornberg, L. D., Perez-Alv erado, G. C., Brothers, P., Pollack, R. M., gr Summers, M. F. (1997) S cience 276, 415-418], both of these resonances disappear in mutants la cking 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 beta 1 and beta 2 protons of Asp-99 to the epsilon protons o f Tyr-14, and from the zeta-OH proton of Tyr-14 to the gamma-carboxyl proton of Asp-99, indicating close proximity of these two residues, an d by other data from the literature. A strong, low-barrier H-bond betw een Asp-99 and Tyr-14 is indicated by the 6.2 ppm deshielding, low fra ctionation factor (phi = 0.34) and slow exchange of the resonance at 1 8.2 ppm, A normal H-bond between Tyr-14 and the steroid is indicated b y the 1.8 ppm deshielding, fractionation factor of 0.97 and the slow e xchange of the resonance at 11.6 ppm. It is suggested that the 10(4.7) -fold contribution of Tyr-14 to catalysis is made possible by strong H -bonding from Asp-99 in the catalytic diad which strengthens general a cid catalysis by Tyr-14. It is also noted that highly deshielded proto n resonances on enzymes between 15 and 20 ppm, assigned to low-barrier H-bonds, generally involve carboxyl groups.