Photophysics of tryptophan fluorescence: Link with the catalytic strategy of the citrate synthase from Thermoplasma acidophilum
Photophysics of tryptophan fluorescence: Link with the catalytic strategy of the citrate synthase from Thermoplasma acidophilum
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DOI：
10.1021/bi048323l
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发表时间：
2005-02-08
期刊：
影响因子：
2.9
通讯作者：
Callis, P
中科院分区：
文献类型：
--
作者：
Kurz, LC;Fite, B;Callis, P
由嗜酸热原体柠檬酸合酶催化的反应中所有主要中间体的形成都伴随着色氨酸荧光的变化。最大的变化是在与底物草酰乙酸（OAA）形成二元配合物时观察到的强猝灭。存在于酶中的四种色氨酸残基以各种组合方式改变为非荧光残基，而不会对蛋白质稳定性、酶机制或其他物理性质造成重大干扰。W348位于距离OAA约9埃的活性位点壁后面的蛋白质疏水核心，负责蛋白质的大部分固有荧光和伴随OAA结合的所有猝灭。寿命研究表明，所有的淬火都是由激发态过程引起的。缺乏溶剂同位素对量子产率的影响排除了涉及质子转移到受体的猝灭机制。当OAA结合时，W348附近残基的单位点突变体改变构象和/或相互作用，其荧光性质没有明显变化。这个结果排除了这些直接角色的变化。电子从吲哚激发态转移到某些受体是主要的猝灭机制；在5f - w取代蛋白中观察到的减少猝灭强化了这一结论。利用无配体酶及其OAA二元配合物的x射线结构，混合量子力学-分子动力学（QM-MM）计算表明，OAA本身是最可能的猝灭剂，OAA羰基作为电子受体。这一结论得到了α -酮酸模型化合物三甲基丙酮酸酯在溶液中作为吲哚荧光扩散猝灭剂的能力的加强。理论计算进一步表明，酶活性位点内OAA羰基周围的正静电势对其接受来自W348激发态的电子的能力至关重要。这些相同的环境因素在激活OAA与乙酰辅酶a碳离子反应中起主要作用。由于羰基极化在许多酶的催化策略中起作用，这些酶的反应涉及到这个官能团，色氨酸荧光的变化可能是其他系统的有用的机制探针。
The formation of all major intermediates in the reaction catalyzed by the citrate synthase from Thermoplasma acidophilum is accompanied by changes in tryptophan fluorescence. The largest change is the strong quenching observed on formation of the binary complex with substrate, oxaloacetate (OAA). The four tryptophan residues present in the enzyme have been changed to nonfluorescent ones in various combinations without major perturbations in protein stability, enzyme mechanism, or other physical properties. W348, residing in the hydrophobic core of the protein behind the active site wall ca. 9 Angstrom from OAA, is responsible for the majority of the protein's intrinsic fluorescence and all of the quenching that accompanies OAA binding. Lifetime studies show that all of the quenching results from excited-state processes. The lack of solvent isotope effects on the quantum yields excludes a quenching mechanism involving proton transfer to an acceptor. There are no significant changes in fluorescence properties in single site mutants of residues near W348 that change conformation and/or interactions when OAA binds. This result excludes these changes from a direct role. Electron transfer from the indole excited state to some acceptor is the major quenching mechanism; the reduced quenching observed in the 5F-W-substituted protein strengthens this conclusion. Using the X-ray structures of the unliganded enzyme and its OAA binary complex, hybrid quantum mechanics-molecular dynamics (QM-MM) calculations show that OAA itself is the most likely quencher with the OAA carbonyl as the electron acceptor. This conclusion is strengthened by the ability of an alpha-keto acid model compound, trimethylpyruvate, to act as a diffusional quencher of indole fluorescence in solution. The theoretical calculations further indicate that the positive electrostatic potential surrounding the OAA carbonyl within the enzymes' active site is essential to its ability to accept an electron from the excited state of W348. These same environmental factors play a major role in activating, OAA to react with the carbanion of acetyl-CoA. Since carbonyl polarization plays a role in the catalytic strategies of numerous enzymes whose reactions involve this functional group, tryptophan fluorescence changes might be useful as a mechanistic probe for other systems.