ELECTROSTATIC AND STERIC CONTRIBUTIONS TO REGULATION AT THE ACTIVE-SITE OF ISOCITRATE DEHYDROGENASE
ELECTROSTATIC AND STERIC CONTRIBUTIONS TO REGULATION AT THE ACTIVE-SITE OF ISOCITRATE DEHYDROGENASE
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DOI：
10.1126/science.2204110
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发表时间：
1990-08-31
期刊：
影响因子：
56.9
通讯作者：
KOSHLAND, DE
中科院分区：
文献类型：
--
作者：
DEAN, AM;KOSHLAND, DE
大肠杆菌的异柠檬酸脱氢酶受活性位点共价修饰的调节，而不是像预期的那样受变构位点的调节。作为评价调节机制的一种手段，比较了底物2 R，3S-异柠檬酸盐和底物类似物2 R-苹果酸盐在天然酶、磷酸化酶和突变酶中的动力学。磷酸化使真实底物的活性降低超过106倍，但引起底物类似物活性的微小变化。动力学结果表明，磷酰基部分和γ-氨基之间的静电排斥和空间位阻是相互作用的。2 R，3S-异柠檬酸酯的羧基是失活的主要原因，而氢键的损失贡献较小。
The isocitrate dehydrogenase of Escherichia coli is regulated by covalent modification at the active site rather than, as expected, at an allosteric site. As a means of evaluating the mechanism of regulation, the kinetics of the substrate, 2R,3S-isocitrate, and a substrate analog, 2R-malate, were compared for the native, phosphorylated, and mutant enzymes. Phosphorylation decreases activity by more than a factor of 106 for the true substrate, but causes minor changes in the activity of the substrate analog. The kinetic results indicate that electrostatic repulsion and steric hindrance between the phosphoryl moiety and the .gamma. carboxyl group of 2R,3S-isocitrate are the major causes of the inactivation, with a lesser contribution from the loss of a hydrogen bond.