Backbone and side chain resonances of steroid-bound delta5-3-ketosteroid isomerase (EC 5.3.3.1), a homodimeric enzyme with 125 residues per monomer, have been assigned by heteronuclear NMR methods with the 15N- and 13C-labeled enzyme. The secondary structure in solution of steroid-bound isomerase, based on interproton NOE's and differences in chemical shifts of backbone H alpha, C alpha, C beta, and CO resonances from random coil values, consists of two alpha-helices (residues 5-21, 48-60), one 3(10) helix (residues 23-30), seven beta-strands (residues 34-38, 44-47, 62-67, 71-73, 78-87, 92-104, and 111-116), and five turns (residues 39-42, 74-77, 88-91, 105-108, and 119-122). Thus isomerase consists of 30% helix, 38% beta-sheet, and 16% turns. The remaining 20 residues (16%) are assumed to form coils. With the exception of a parallel interaction between beta-strands 1 and 7, all beta-strand interactions are antiparallel, forming both a beta-hairpin (beta1, beta2) and a four-stranded beta-sheet in which the first strand is interrupted (beta3-beta4, beta5, beta6, beta7). 1H-15N HSQC titrations of the free enzyme with the substrate analog 19-nortestosterone hemisuccinate revealed steroid-induced changes in backbone 15N and NH chemical shifts throughout the enzyme, with maximal effects on helix I (Val-15), beta-strand 1 of the beta-hairpin (Asp-38), the loop between helix 3 and beta-strand 3 (Leu-61), beta-strand 3 (Ala-64), beta-strand 5 (Phe-82, Ser-85, Glu-87), beta-strand 6 (Ile-98), and beta-strand 7 (Ala-114, Phe-116) of the beta-sheet, thus indicating the secondary structural components involved in steroid binding. These effects include regions near the catalytic residues Tyr-14 and Asp-38 which function as the general acid and base, respectively, in the ketosteroid isomerase reaction. Intermolecular NOE's between 19-nortestosterone hemisuccinate and isomerase indicate that the steroid binds near alpha-helices 1 and 3, which form one wall of the active site, and one end of the four-stranded beta-sheet which forms the other wall. Consistent with these observations, doxyldihydrotestosterone, a steroid that is spin-labeled at its solvent-exposed end [Kuliopulos, A., Westbrook, E. M., Talalay, P., & Mildvan, A. S. (1987) Biochemistry 26, 3927-3937], induced the selective attenuation in the 1H-15N HSQC spectra of cross peaks of residues at the end of helix 3 (Ser-58, Leu-59, Lys-60, Leu-61), beta-strand 5 (Val-84, Ser-85), and beta-strand 6 (Val-95), due to the proximity of the nitroxide radical to the backbone 15N and NH nuclei of these residues, thus confirming the location of the D ring of the bound steroid and defining the mouth of the active site.
Read full abstract