title: 13C NMR relaxation studies of backbone and side chain motion of the catalytic tyrosine residue in free and steroid-bound delta 5-3-ketosteroid isomerase.
abstract: Side chain and backbone dynamics of the catalytic residue, Tyr-14, in free and steroid-bound delta 5-3-ketosteroid isomerase (EC 5.3.3.1, homodimer, M(r) = 26.8 kDa) have been examined by measurements of longitudinal and transverse 13C relaxation rates and nuclear Overhauser effects at both 500 and 600 MHz (proton frequencies). The data, analyzed using the model-free formalism, yielded an optimized correlation time for overall molecular rotation (tau m) of 17.9 ns, in agreement with the result (18 ns) of fluorescence anisotropy decay measurements [Wu, P., Li, Y.-K., Talalay, P., & Brand, L. (1994) Biochemistry 33, 7415-7422] and Stokes' law calculation (20 ns). The order parameter of the side chain C epsilon of Tyr-14 (S2 = 0.74), which is a measure of the restriction of its high-frequency (nanosecond to picosecond) motion, was significantly lower than that of the backbone C alpha (S2 = 0.82), indicating greater restriction of backbone motion. Upon binding of the steroid ligand, 19-nortestosterone hemisuccinate, a product analog and substrate of the reverse isomerase reaction, S2 of the side chain C epsilon increased from 0.74 to 0.86, while that of the backbone C alpha did not change significantly. Thus, in the steroid complex, the amplitude of high-frequency side chain motion of Tyr-14 became more restricted than that of its backbone which could lower the entropy barrier to catalysis. Lower-frequency (millisecond to microsecond) motion of Tyr-14 at rates comparable to kcat were detected by exchange contributions to transverse relaxation of both C epsilon and C alpha. Steroid binding produced no change in this low-frequency motion of the side chain of Tyr-14, which could contribute to substrate binding and product release, but decreased the exchange contribution to transverse relaxation of the backbone.
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Zhao
authors.forename: C
authors.initials: C
authors.lastname: Abeygunawardana
authors.forename: A S
authors.initials: AS
authors.lastname: Mildvan
mesh_terms: D000735:Androstenedione / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002247:Carbon Isotopes; D003198:Computer Simulation; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009010:Monte Carlo Method; D009038:Motion; D013251:Steroid Isomerases / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
chemical_list: D002247:Carbon Isotopes; D000735:Androstenedione; D014443:Tyrosine; D013251:Steroid Isomerases; C022660:steroid delta-isomerase