Evidence for a functional role of the dynamics of glycine-121 of Escherichia coli dihydrofolate reductase obtained from kinetic analysis of a site-directed mutant
Evidence for a functional role of the dynamics of glycine-121 of Escherichia coli dihydrofolate reductase obtained from kinetic analysis of a site-directed mutant
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DOI：
10.1021/bi9716231
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发表时间：
1997-12-16
期刊：
影响因子：
2.9
通讯作者：
Benkovic, SJ
中科院分区：
文献类型：
--
作者：
Cameron, CE;Benkovic, SJ
来自大肠杆菌的二氢叶酸还原酶（DHFR）的二维杂合（H-1-N-15）核磁弛豫研究已经证明，距离酶的催化中心19埃的甘氨酸-121具有纳秒时间尺度上的大幅度骨架运动[Epstein，D. M.，Benkovic，S. J.，和Wright，P.E.（1995）Biochemistry 34，11037-11048]。为了探索该残基的动力学-功能关系，我们构建了一种突变酶，其中该甘氨酸被改变为缬氨酸。平衡结合研究表明，瓦尔-121突变体保留了野生型与二氢叶酸和四氢叶酸的结合特性;然而，与NADPH和NADP（+）的结合相对于野生型DHFR分别降低了40倍和2倍。单次转换实验表明，氢化物转移减少了200倍，速率为1.3 s（-1），是稳态下的限速步骤。有趣的是，对瓦尔-121突变体进行的稳态前动力学分析显示，在化学反应之前发生了构象变化，其速率为3.5 s（-1）。如果这一步存在于野生型酶的动力学机制中，那么预计它将以大约2000 s（-1）的速率发生。甘氨酸-121也变为丙氨酸、丝氨酸、亮氨酸和脯氨酸。虽然Ala-121和Ser-121突变体的行为与野生型DHFR相似，但Leu-121和Pro-121突变体的行为与瓦尔-121 DHFR相似，因为氢化物转移是稳态下的限速步骤，并且观察到化学之前的构象变化。最后，在氨基酸121和122之间插入甘氨酸或缬氨酸，分别产生具有与野生型或瓦尔-121 DHF相似性质的突变酶。两者合计，这些结果提供了令人信服的证据动态耦合的远程残基动力学事件的活性位点的DHFR。
Two-dimensional heteronuclear (H-1-N-15) nuclear magnetic relaxation studies of dihydrofolate reductase (DHFR) from Escherichia coli have demonstrated that glycine-121 which is 19 Angstrom from the catalytic center of the enzyme has large-amplitude backbone motions on the nanosecond time scale [Epstein, D. M., Benkovic, S. J., and Wright, P. E. (1995) Biochemistry 34, 11037-11048]. in order to probe the dynamic-function relationships of this residue, we constructed a mutant enzyme in which this glycine was changed to valine. Equilibrium binding studies indicated that the Val-121 mutant retained wild-type binding properties with respect to dihydrofolate and tetrahydrofolate; however, binding to NADPH and NADP(+) was decreased by 40-fold and 2-fold, respectively, relative to wild-type DHFR. Single-turnover experiments indicated that hydride transfer was reduced by 200-fold to a rate of 1.3 s(-1) and was the rate-limiting step in the steady state. Interestingly, pre-steady-state kinetic analysis of the Val-121 mutant revealed a conformational change which preceded chemistry that occurred at a rate of 3.5 s(-1). If this step exists in the kinetic mechanism of the wild-type enzyme, then it would be predicted to occur at a rate of approximately 2000 s(-1). Glycine-121 was also changed to alanine, serine, leucine, and proline. While the Ala-121 and Ser-121 mutants behaved similar to wild-type DHFR, the Leu-121 and Pro-121 mutants behaved like Val-121 DHFR in that hydride transfer was the rate-limiting step in the steady state and a conformational change preceding chemistry was observed. Finally, insertion of a glycine or valine between amino acids 121 and 122 produced mutant enzymes with properties similar to wild-type or Val-121 DHFRs, respectively. Taken together, these results provide compelling evidence for dynamic coupling of a remote residue to kinetic events at the active site of DHFR.