Determinants of performance in the isocitrate dehydrogenase of Escherichia coli.
Determinants of performance in the isocitrate dehydrogenase of Escherichia coli.
复制标题
大肠杆菌异柠檬酸脱氢酶性能的决定因素。
DOI：
10.1002/pro.5560050218
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
KoshlandJr,DE
中科院分区：
文献类型：
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作者：
Dean,AM;Shiau,AK;KoshlandJr,DE
通过结合定点诱变和替代底物的利用，研究了大肠杆菌 NADP 依赖性异柠檬酸脱氢酶的底物特异性。野生型酶与 2R-苹果酸的动力学比较表明，2R、3S-异柠檬酸的 γ-羧酸盐对酶性能的影响为 12, 000, 000。对 10 种酶和 9 种不同底物的动力学数据分析表明，1, 650 的因子可归因于 S113 和结合异柠檬酸的 γ-羧酸盐之间形成的氢键，150 的因子可归因于 γ-羧酸盐的负电荷，50 的因子可归因于 γ-甲基。这些结果与米氏配合物的 X 射线结构完全一致，该结构显示氢键定位异柠檬酸的 γ-羧酸盐，从而可以与 NADP 的烟酰胺环形成盐桥。
The substrate specificity of the NADP‐dependent isocitrate dehydrogenase ofEscherichia coliwas investigated by combining site‐directed mutagenesis and utilization of alternative substrates. A comparison of the kinetics of the wild‐type enzyme with 2R‐malate reveals that the γ‐carboxylate of 2R, 3S‐isocitrate contributes a factor of 12, 000, 000 to enzyme performance. Analysis of kinetic data compiled for 10 enzymes and nine different substrates reveals that a factor of 1, 650 can be ascribed to the hydrogen bond formed between S113 and the γ‐carboxylate of bound isocitrate, a factor of 150 to the negative charge of the γ‐carboxylate, and a factor of 50 for the γ‐methyl. These results are entirely consistent with X‐ray structures of Michaelis complexes that show a hydrogen bond positions the γ‐carboxylate of isocitrate so that a salt bridge can form to the nicotinamide ring of NADP.