Ohmae E, Iriyama K, Ichihara S, Gekko K
Department of Applied Biological Science, Faculty of Agriculture, Nagoya University.
J Biochem. 1996 Apr;119(4):703-10. doi: 10.1093/oxfordjournals.jbchem.a021299.
To elucidate the role of a flexible loop (residues 64-72) in the stability and function of Escherichia coli dihydrofolate reductase, glycine-67 in this loop was substituted by site-directed mutagenesis with seven amino acids (Ala, Cys, Asp, Leu, Ser, Thr, and Val). The circular dichroism spectra suggested that the confirmation of the native structure was affected by the mutations in both the presence and absence of NADPH. The free energy change of unfolding by urea decreased in the order of G67A > G67S > or = wild-type > or = G67D > G67T > G67C > or = G67L > G67V. The steady-state kinetic parameters for the enzyme reaction, Km and kcat, were only slightly influenced, but the rate of the hydride transfer reaction was significantly changed by the mutations, as revealed by the deuterium isotope effect on the enzyme activity. These results suggest that site 67 in the flexible loop, being very far from the active site, plays an important role in the stability and function of this enzyme. The characteristics of the mutations were discussed in terms of the modified flexibility of the native structure, compared with the results of mutations at site 121 in another flexible loop.
为阐明柔性环（64 - 72位氨基酸残基）在大肠杆菌二氢叶酸还原酶稳定性和功能中的作用，通过定点诱变将该环中的甘氨酸67替换为7种氨基酸（丙氨酸、半胱氨酸、天冬氨酸、亮氨酸、丝氨酸、苏氨酸和缬氨酸）。圆二色光谱表明，无论有无NADPH，天然结构的构象均受这些突变的影响。尿素诱导的去折叠自由能变化按以下顺序降低：G67A > G67S > 或 = 野生型 > 或 = G67D > G67T > G67C > 或 = G67L > G67V。酶反应的稳态动力学参数Km和kcat仅受到轻微影响，但氘同位素效应显示，这些突变显著改变了氢化物转移反应的速率。这些结果表明，柔性环中的67位氨基酸残基虽距活性位点很远，但对该酶的稳定性和功能起着重要作用。与另一个柔性环中121位氨基酸残基的突变结果相比，根据天然结构修饰后的柔韧性对这些突变的特征进行了讨论。
