Schnell Jason R, Dyson H Jane, Wright Peter E
Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Biochemistry. 2004 Jan 20;43(2):374-83. doi: 10.1021/bi035464z.
Dihydrofolate reductase (DHFR) has several flexible active site loops that facilitate ligand binding and catalysis. Previous studies of backbone dynamics in several complexes of DHFR indicate that the time scale and amplitude of motion depend on the conformation of the active site loops. In this study, information on dynamics is extended to methyl-containing side chains. To understand the role of side chain dynamics in ligand binding and loop conformation, methyl deuterium relaxation rates of Escherichia coli DHFR in binary folate and ternary folate:NADP+ complexes have been measured, together with chi(1) rotamer populations for threonine, isoleucine, and valine residues, determined from measurements of 3J(CgammaCO) and 3J(CgammaN) coupling constants. The results indicate that, in addition to backbone motional restriction in the adenosine-binding site, side chain flexibility in the active site and the surrounding active site loops is diminished upon binding NADP+. Resonances for several methyls in the active site and the surrounding active site loops were severely broadened in the folate:NADP+ ternary complex, suggesting the presence of motion on the chemical shift time scale. The side chains of Ile14 and Ile94, which pack against the nicotinamide and pterin rings of the cofactor and substrate, respectively, exhibit rotamer disorder in the ternary folate:NADP+ complex. Conformational fluctuations of these side chains may play a role in transition state stabilization; the observed line broadening for Ile14 suggests motions on a microsecond/millisecond time scale.
二氢叶酸还原酶（DHFR）有几个灵活的活性位点环，有助于配体结合和催化作用。先前对DHFR几种复合物主链动力学的研究表明，运动的时间尺度和幅度取决于活性位点环的构象。在本研究中，动力学信息扩展到含甲基的侧链。为了了解侧链动力学在配体结合和环构象中的作用，已测量了大肠杆菌DHFR在二元叶酸和三元叶酸：NADP +复合物中的甲基氘弛豫率，以及从3J（CγCO）和3J（CγN）耦合常数测量中确定的苏氨酸、异亮氨酸和缬氨酸残基的χ(1)旋转异构体群体。结果表明，除了腺苷结合位点的主链运动受限外，结合NADP +后活性位点及周围活性位点环中的侧链灵活性降低。在叶酸：NADP +三元复合物中，活性位点及周围活性位点环中几个甲基的共振严重变宽，表明在化学位移时间尺度上存在运动。分别与辅因子和底物的烟酰胺环和蝶呤环堆积的Ile14和Ile94的侧链，在三元叶酸：NADP +复合物中表现出旋转异构体无序。这些侧链的构象波动可能在过渡态稳定中起作用；观察到的Ile14谱线展宽表明在微秒/毫秒时间尺度上存在运动。
