NMR-STUDIES OF THE SECONDARY STRUCTURE IN SOLUTION AND THE STEROID-BINDING SITE OF DELTA(5)-3-KEROSTEROID ISOMERASE IN COMPLEXES WITH DIAMAGNETIC AND PARAMAGNETIC STEROIDS

Citation
Qj. Zhao et al., NMR-STUDIES OF THE SECONDARY STRUCTURE IN SOLUTION AND THE STEROID-BINDING SITE OF DELTA(5)-3-KEROSTEROID ISOMERASE IN COMPLEXES WITH DIAMAGNETIC AND PARAMAGNETIC STEROIDS, Biochemistry, 36(12), 1997, pp. 3458-3472
Citations number
64
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
36
Issue
12
Year of publication
1997
Pages
3458 - 3472
Database
ISI
SICI code
0006-2960(1997)36:12<3458:NOTSSI>2.0.ZU;2-D
Abstract
Backbone and side chain resonances of steroid-bound Delta(5)-3-ketoste roid isomerase (EC 5.3.3.1), a homodimeric enzyme with 125 residues pe r monomer, have been assigned by heteronuclear NMR methods with the N- 15- and C-13-labeled enzyme. The secondary structure in solution of st eroid-bound isomerase, based on interproton NOE's and differences in c hemical shifts of backbone H alpha, C alpha, C beta, and CO resonances from random coil values, consists of two cl-helices (residues 5-21, 4 8-60), 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 turn s (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 2 0 residues (16%) are assumed to form coils. With the exception of a pa rallel interaction between beta-strands 1 and 7, all beta-strand inter actions are antiparallel, forming both a beta-hairpin (beta 1, beta 2) and a four-stranded beta-sheet in which the first strand is interrupt ed (beta 3-beta 4, beta 5, beta 6, beta 7). H-1-N-15 HSQC titrations o f the free enzyme with the substrate analog 19-nortestosterone hemisuc cinate revealed steroid-induced changes in backbone N-15 and NH chemic al shifts throughout the enzyme, with maximal effects on helix 1 (Val- 15), beta-strand 1 of the beta-hairpin (Asp-38), the loop between heli x 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 (A la-114, Phe-116) of the beta-sheet, thus indicating the secondary stru ctural components involved in steroid binding. These effects include r egions 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 hemisuccina te and isomerase indicate that the steroid binds near cx-helices 1 and 3, which form one wall of the active site, and one end of the four-st randed beta-sheet which forms the other wall. Consistent with these ob servations, doxyldihydrotestosterone, a steroid that is spin-labeled a t its solvent-exposed end [Kuliopulos, A., Westbrook, E. M., Talalay, P., & Mildvan, A. S. (1987) Biochemistry 26, 3927-3937], induced the s elective attenuation in the H-1-N-15 HSQC spectra of cross peaks of re sidues at the end of helix 3 (Ser-58, Leu-59, Lys-60, Leu-61), beta-st rand 5 (Val-84, Ser-85), and beta-strand 6 (Val-95), due to the proxim ity of the nitroxide radical to the backbone N-15 and NH nuclei of the se residues, thus confirming the location of the D ring of the bound s teroid and defining the mouth of the active site.