Zhao Q, Abeygunawardana C, Mildvan A S
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Biochemistry. 1996 Feb 6;35(5):1525-32. doi: 10.1021/bi9525381.
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.
通过在500和600 MHz（质子频率）下测量纵向和横向(^{13}C)弛豫率以及核Overhauser效应，研究了游离和甾体结合的δ5-3-酮甾体异构酶（EC 5.3.3.1，同二聚体，(M(r)) = 26.8 kDa）中催化残基Tyr-14的侧链和主链动力学。使用无模型形式分析数据，得出整体分子旋转的优化相关时间（(\tau_m)）为17.9 ns，与荧光各向异性衰减测量结果（18 ns）[Wu, P., Li, Y.-K., Talalay, P., & Brand, L. (1994) Biochemistry 33, 7415 - 7422]以及斯托克斯定律计算结果（20 ns）一致。Tyr-14侧链(C\epsilon)的序参数（(S_2 = 0.74)），它是其高频（纳秒到皮秒）运动受限程度的一种度量，显著低于主链(C\alpha)的序参数（(S_2 = 0.82)），表明主链运动受限程度更大。在甾体配体19-去甲睾酮半琥珀酸酯（一种反向异构酶反应的产物类似物和底物）结合后，侧链(C\epsilon)的(S_2)从0.74增加到0.86，而主链(C\alpha)的(S_2)没有显著变化。因此，在甾体复合物中，Tyr-14高频侧链运动的幅度比其主链运动的幅度受到更多限制，这可能会降低催化的熵垒。通过对(C\epsilon)和(C\alpha)横向弛豫的交换贡献，检测到Tyr-14以与(k_{cat})相当的速率进行的低频（毫秒到微秒）运动。甾体结合对Tyr-14侧链的这种低频运动没有影响，这可能有助于底物结合和产物释放，但降低了对主链横向弛豫的交换贡献。
