STEREOCHEMISTRY OF THE CONCERTED ENOLIZATION CATALYZED BY DELTA-5-3-KETOSTEROID ISOMERASE

The reaction catalyzed by DELTA-5-3-ketosteroid isomerase has been shown to occur via the concerted enolization of the DELTA-5-3-ketosteroid substrate to form a dienolic intermediate, brought about by Tyr-14, which hydrogen bonds to and protonates the 3-keto group, and Asp-38, which removes and axial (beta) proton from C-4 of the substrate, in the same rate-limiting step [Xue, L., Talalay, P., & Mildvan, A. S. (1990) Biochemistry 29, 7491-7500; Kuliopulos, A., Mildvan, A. S., Shortle, D., & Talalay, P. (1989) Biochemistry 26, 3927-3937]. Since the axial C-4 proton is removed by Asp-38 from above the substrate, a determination of the complete stereochemistry of this rapid, concerted enolization requires information on the direction of approach of Tyr-14 to the enzyme-bound steroid. The double mutant enzyme, Y55F + Y88F, which retains Tyr-14 as the sole Tyr residue, was prepared and showed only a 4.5-fold decrease in k(cat) (12 000 s-1) and a 3.6-fold decrease in K(M) (94-mu-M) for DELTA-5-androstene-3,17-dione, in comparison with the wild-type enzyme. Deuteration of the aromatic rings of the 10 Phe residues further facilitated the assignment of the aromatic proton resonances of Tyr-14 in the 600-MHz TOCSY spectrum at 6.66 +/- 0.01 ppm (3,5H) and at 6.82 +/- 0.01 ppm (2,6H). Variation of the pH from 4.9 to 10.9 did not alter these shifts, indicating that the pK(a) of Tyr-14 exceeds 10.9. Resonances assigned to the three His residues titrated with pK(a) values very similar to those found with the wild-type enzyme. The binding of 19-nortestosterone, a product analogue and substrate of the reverse isomerase reaction, induced downfield shifts of -0.12 and -0.06 ppm of the 3,5- and 2,6-proton resonances of Tyr-14, respectively, possibly due to deshielding by the 3-keto group of the steroid, but also induced +0.29 to -0.41 ppm changes in the chemical shifts of 8 of the 10 Phe residues and smaller changes in 10 of the 12 ring-shifted methyl resonances, indicating a steroid-induced conformation change in the enzyme. NOESY spectra in H2O revealed strong negative Overhauser effects from the 3,5-proton resonance of Tyr-14 to the overlapping 2-alpha, 2-beta, or 6-beta-proton resonances of the bound steroid but no NOE's to the 4- or 6-alpha-protons of the steroid. These observations indicate the NOE's to be from Tyr-14 to the 2-alpha- and 2-beta-protons rather than to the 6-beta-proton of the steroid. Prolonged incubation of the enzyme-steroid complex in (H2O)-H-2 resulted in deuteration of the 6-beta-, 4-, and 2-alpha-positions of the steroid. The remaining 2-beta-proton retained 13% of the original NOE from Tyr-14, indicating that 87% of the NOE was to the 2-alpha-proton. These observations are consistent with a specific and asymmetric orientation of Tyr-14 with respect to the bound steroid that places to phenolic ring of Tyr-14 beside C-2 and far from C-4 and C-6 of the steroid. This orientation permits Tyr-14 to form a hydrogen bond to the lone electron pair of the carbonyl oxygen of the steroid product that is trans to the C-4-C-5 double bond. On the basis of the geometry of this product complex and the fact that the axial C-4 beta-proton of the substrate is removed by Asp-38 from above the steroid, we conclude that an orthogonal (rather than suprafacial or antarafacial) arrangement of the proton donor and proton acceptor, with respect to the bound substrate, is stereoelectronically appropriate for a rapid, concerted enolization.