C-13 NMR RELAXATION STUDIES OF BACKBONE AND SIDE-CHAIN MOTION OF THE CATALYTIC TYROSINE RESIDUE IN FREE AND STEROID-BOUND DELTA(5)-3-KETOSTEROID ISOMERASE

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, h omodimer, M(r) = 26.8 kDa) have been examined by measurements of longi tudinal and transverse C-13 relaxation rates and nuclear Overhauser ef fects 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 (S-2 = 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 (S-2 = 0.82) , indicating greater restriction of backbone motion. Upon binding of t he steroid ligand, 19-nortestosterone hemisuccinate, a product analog and substrate of the reverse isomerase reaction, S-2 Of the side chain C-epsilon increased from 0.74 to 0.86, while that of the backbone C-a lpha did not change significantly. Thus, in the steroid complex, the a mplitude of high-frequency side chain motion of Tyr-14 became more res tricted than that of its backbone which could lower the entropy barrie r to catalysis. Lower-frequency (millisecond to microsecond) motion of Tyr-14 at rates comparable to k(cat) were detected by exchange contri butions to transverse relaxation of both C-epsilon and C-alpha. Steroi d binding produced no change in this low-frequency motion of the side chain of Tyr-14, which could contribute to substrate binding and produ ct release, but decreased the exchange contribution to transverse rela xation of the backbone.