Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase.
Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase.
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DOI：
10.1021/jacs.5b09328
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发表时间：
2015-12-09
影响因子：
15
通讯作者：
Richard JP
中科院分区：
文献类型：
--
作者：
Zhai X;Amyes TL;Richard JP
磷酸三糖异构酶 (TIM) 第 7 环的 Y208 和 S211 侧链与第 6 环的骨架酰胺和羰基形成氢键，以稳定笼状酶-底物复合物。七个突变 [Y208T、Y208S、Y208A、Y208F、S211G、S211A、Y208T/S211G] 对整个底物磷酸二羟丙酮和 d-甘油醛 3-磷酸 [(kcat/Km)GAP 和 (kcat/Km)DHAP] 以及底物碎片的 TIM 催化反应动力学参数的影响报道了乙醇醛和亚磷酸二价阴离子 (kcat/KHPiKGA)。这些动力学参数之间的线性对数相关性（斜率为1.04±0.03）表明，大多数TIM突变会导致整个底物和底物片段的催化反应的活化势垒发生相同的变化，从而通过与蛋白质催化剂的类似相互作用来稳定这些反应的过渡态。 GAP 异构化的 kcat 和亚磷酸双阴离子与底物片段的过渡态结合的 Kd⧧ 之间的第二个线性对数相关性 [斜率 = 0.53 ± 0.16] 显示约。 50% 的野生型 TIM 二价阴离子结合能被这些突变消除，在野生型米氏复合体上表达，大约 50% 的二价阴离子结合能在野生型米氏复合体上表达。 50%仅在野生型过渡态下表达。当突变导致催化位点酶反应性降低时，就会观察到与这种相关性的负偏差。 Y208T、Y208S 和 Y208A 突变的主要影响是导致总内在二价阴离子结合能降低，但 Y208F 的影响延伸到催化位点。
The side chains of Y208 and S211 from loop 7 of triosephosphate isomerase (TIM) form hydrogen bonds to backbone amides and carbonyls from loop 6 to stabilize the caged enzyme–substrate complex. The effect of seven mutations [Y208T, Y208S, Y208A, Y208F, S211G, S211A, Y208T/S211G] on the kinetic parameters for TIM catalyzed reactions of the whole substrates dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate [(kcat/Km)GAP and (kcat/Km)DHAP] and of the substrate pieces glycolaldehyde and phosphite dianion (kcat/KHPiKGA) are reported. The linear logarithmic correlation between these kinetic parameters, with slope of 1.04 ± 0.03, shows that most mutations of TIM result in an identical change in the activation barriers for the catalyzed reactions of whole substrate and substrate pieces, so that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The second linear logarithmic correlation [slope = 0.53 ± 0.16] between kcat for isomerization of GAP and Kd⧧ for phosphite dianion binding to the transition state for wildtype and many mutant TIM-catalyzed reactions of substrate pieces shows that ca. 50% of the wildtype TIM dianion binding energy, eliminated by these mutations, is expressed at the wildtype Michaelis complex, and ca. 50% is only expressed at the wildtype transition state. Negative deviations from this correlation are observed when the mutation results in a decrease in enzyme reactivity at the catalytic site. The main effect of Y208T, Y208S, and Y208A mutations is to cause a reduction in the total intrinsic dianion binding energy, but the effect of Y208F extends to the catalytic site.