DYNAMICS OF A FLEXIBLE LOOP IN DIHYDROFOLATE-REDUCTASE FROM ESCHERICHIA-COLI AND ITS IMPLICATION FOR CATALYSIS
DYNAMICS OF A FLEXIBLE LOOP IN DIHYDROFOLATE-REDUCTASE FROM ESCHERICHIA-COLI AND ITS IMPLICATION FOR CATALYSIS
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DOI：
10.1021/bi00168a007
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发表时间：
1994-01-18
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：
FALZONE, CJ;WRIGHT, PE;BENKOVIC, SJ
在室温下，来自大肠杆菌的脱氢叶酸还原酶在核磁共振时间尺度上缓慢地采集了两个不同的环境。几个指定的共振属于由残基9-24组成的环(环1)中或其附近的残基。通过从环上删除形成残基的三个发夹转角并用单个甘氨酸填充缺口来改变这种交换过程(或者移除或在核磁共振时间尺度上变快)[Li，L.，Falzone，C.J.，Wright，P.E.，&Benkovic，S.J.(I 992)BioChemical 31,7826-7833]。在二维核Overhauser谱中，通过分析Trp-22的N(Epsilon)H的交叉峰体积随时间的变化，得到apo-DHFR中与环I有关的交换率的近似值35 S-1，该残基位于该环中并已分解了交叉峰。由于该环在催化中起着关键作用，四氢叶酸的构象交换和关闭速率与产物和底物复合体中烟酰胺辅因子的减少之间的对应关系表明，环的移动可能是底物周转的限制因素。
Apo-dihydrofolate reductase from Escherichia coli samples two distinct environments slowly on the NMR time scale at room temperature. Several assigned resonances belong to residues in, or proximal to, a loop (loop 1) which is comprised of residues 9-24. This exchange process was altered (either removed or made fast on the NMR time scale) by deleting three hairpin turn forming residues from the loop and filling the gap with a single glycine [Li, L., Falzone, C. J., Wright, P. E., & Benkovic, S. J. (I 992) Biochemistry 31, 7826-7833]. An approximate value of 35 s-1 for the exchange rate associated with loop I in apo-DHFR was obtained in two-dimensional nuclear Overhauser spectra by analyzing the time dependence of the cross-peak volume for N(epsilon)H of Trp-22, a residue which is located in this loop and which has resolved cross-peaks. Owing to the critical role that this loop plays in catalysis, the correspondence between this rate of conformational exchange and off-rates for tetrahydrofolate and the reduced nicotinamide cofactor from product and substrate complexes suggests that loop movement may be a limiting factor in substrate turnover.