Zhang H C, Cisneros R J, Deng W L, Johnson L F, Dunlap R B
Department of Biochemistry, Ohio State University, Columbus 43210.
Biochem Biophys Res Commun. 1990 Mar 30;167(3):869-75. doi: 10.1016/0006-291x(90)90604-l.
The arginine located at position 44 of mouse thymidylate synthase is in a highly conserved loop that is in close proximity to the active site cleft of the enzyme. Structural analyses have suggested that this arginine forms hydrogen bonds with the alpha-carboxylate of the C-terminal amino acid and the phosphate of the substrate analog, FdUMP (D. A. Matthews, K. Appelt and S. J. Oatley, (1989) Adv. Enz. Reg., 29, 47-60). We have used protein engineering techniques to change this amino acid residue to valine. This alteration leads to large reductions in the ability of the enzyme to form covalent complexes with substrate (dUMP) or inhibitor (FdUMP) and at least a 100-fold reduction in catalytic activity. These observations show that this arginine plays an important role in maintaining catalytic activity of the enzyme.
小鼠胸苷酸合成酶第44位的精氨酸位于一个高度保守的环中，该环紧邻酶的活性位点裂隙。结构分析表明，这个精氨酸与C端氨基酸的α-羧基以及底物类似物FdUMP的磷酸基团形成氢键（D. A. 马修斯、K. 阿佩尔特和S. J. 奥特利，（1989年）《酶学研究进展》，29卷，47 - 60页）。我们运用蛋白质工程技术将这个氨基酸残基替换为缬氨酸。这种改变导致酶与底物（dUMP）或抑制剂（FdUMP）形成共价复合物的能力大幅降低，催化活性至少降低100倍。这些观察结果表明，这个精氨酸在维持酶的催化活性方面起着重要作用。
