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首页> 外文期刊>Biochemistry >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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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

机译：通过定点突变体的动力学分析获得的大肠杆菌二氢叶酸还原酶甘氨酸121动力学动力学的功能性证据

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摘要

Two-dimensional heteronuclear (1H-15N) nuclear magnetic relaxation studies of dihydrofolate reductase (DHFR) from Escherichia coli have demonstrated that glycine-121 which is 19 A 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.
机译：大肠杆菌二氢叶酸还原酶（DHFR）的二维异核（1H-15N）核磁弛豫研究表明，距该酶催化中心19 A的甘氨酸121具有纳秒级的大幅度骨架运动[Epstein，DM，Benkovic，SJ和Wright，PE（1995）Biochemistry 34，11037-11048]。为了探究该残基的动态功能关系，我们构建了一种突变酶，其中该甘氨酸变为缬氨酸。平衡结合研究表明，Val-121突变体保留了相对于二氢叶酸和四氢叶酸的野生型结合特性。然而，相对于野生型DHFR，与NADPH和NADP +的结合分别降低了40倍和2倍。单周转实验表明，氢化物转移减少了200倍，达到1.3 s-1的速率，并且是稳态下的限速步骤。有趣的是，对Val-121突变体的稳态前动力学分析表明，构象变化先于化学反应，发生速率为3.5 s-1。如果此步骤存在于野生型酶的动力学机制中，则可以预测其发生的速率约为2000 s-1。甘氨酸121也更改为丙氨酸，丝氨酸，亮氨酸和脯氨酸。尽管Ala-121和Ser-121突变体的行为类似于野生型DHFR，但Leu-121和Pro-121突变体的行为类似于Val-121 DHFR，因为氢化物转移是稳态下的限速步骤，观察到之前的化学变化。最后，在氨基酸121和122之间插入甘氨酸或缬氨酸产生了突变酶，其特性分别类似于野生型或Val-121 DHFR。综上所述，这些结果提供了令人信服的证据，说明了远端残基与DHFR活性位点的动力学事件发生动态偶联。

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