The role of glutamate 87 in the kinetic mechanism of Thermus thermophilus isopropylmalate dehydrogenase.
The role of glutamate 87 in the kinetic mechanism of Thermus thermophilus isopropylmalate dehydrogenase.
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谷氨酸 87 在嗜热栖热菌异丙基苹果酸脱氢酶动力学机制中的作用。
DOI：
10.1002/pro.5560041022
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Dvorak,L
中科院分区：
文献类型：
--
作者：
Dean,AM;Dvorak,L
研究了嗜热栖热菌NAD依赖的异丙基苹果酸脱氢酶催化2 R，3S-异丙基苹果酸氧化脱羧反应的动力学机制。对于野生型和两种突变酶（E87 G和E87 Q），无论使用的是天然底物还是替代底物（2 R-苹果酸盐、2 R，3S-酒石酸盐和/或NADP），均获得了随机或稳态有序顺序机制的典型初始速率结果。尽管突变和替代底物导致特异性（kcat/Km）显著降低，但初始速率数据未能收敛于快速平衡有序模式。虽然单独的抑制研究可能会建议一个有序的动力学机制与辅因子结合第一，详细的分析表明，预期的非竞争性模式出现竞争性，因为从三元复合物的解离常数远小于那些从二元复合物。平衡荧光研究都证实了随机结合的基板和动力学估计的解离常数的基板从二元复合物。后者不受87位突变的显著干扰。位点87的突变不影响二元复合物的解离常数，但确实大大增加了米氏常数，表明E87有助于稳定野生型酶的米氏复合物。现有的结构数据，动力学结果的模式，以及大肠杆菌同源异柠檬酸脱氢酶的假米氏复合物的结构表明，E87与烟酰胺环相互作用。
The kinetic mechanism of the oxidative decarboxylation of 2R,3S‐isopropylmalate by the NAD‐dependent isopropylmalate dehydrogenase ofThermus thermophiluswas investigated. Initial rate results typical of random or steady‐state ordered sequential mechanisms are obtained for both the wild‐type and two mutant enzymes (E87G and E87Q) regardless of whether natural or alternative substrates (2R‐malate, 2R,3S‐tartrate and/or NADP) are utilized. Initial rate data fail to converge on a rapid equilibrium‐ordered pattern despite marked reductions in specificity (kcat/Km) caused by the mutations and alternative substrates. Although the inhibition studies alone might suggest an ordered kinetic mechanism with cofactor binding first, a detailed analysis reveals that the expected noncompetitive patterns appear uncompetitive because the dissociation constants from the ternary complexes are far smaller than those from the binary complexes. Equilibrium fluorescence studies both confirm the random binding of substrates and the kinetic estimates of the dissociation constants of the substrates from the binary complexes. The latter are not disturbed markedly by the mutations at site 87. Mutations at site 87 do not affect the dissociation constants from the binary complexes, but do greatly increase the Michaelis constants, indicating that E87 helps stabilize the Michaelis complex of the wild‐type enzyme. The available structural data, the patterns of the kinetics results, and the structure of a pseudo‐Michaelis complex of the homologous isocitrate dehydrogenase ofEscherichia colisuggest that E87 interacts with the nicotinamide ring.