Carbon-deuterium bonds as probes of dihydrofolate reductase
Carbon-deuterium bonds as probes of dihydrofolate reductase
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DOI：
10.1021/ja0779607
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发表时间：
2008-05-21
影响因子：
15
通讯作者：
Romesberg, Floyd E.
中科院分区：
文献类型：
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作者：
Thielges, Megan C.;Case, David A.;Romesberg, Floyd E.
人们一直致力于了解静电学和动力学对蛋白质功能的贡献，特别是对酶催化的贡献。不幸的是，这些研究由于缺乏直接的实验探针而受到限制，我们一直在开发利用碳-重键作为蛋白质的探针，现在报告了该技术在催化氢化物转移的CLHB酶中的应用，该酶已成为生物催化的范例。我们观察到(甲基-d(3))甲硫氨酸碳-氘键的伸缩吸收频率与溶剂介电常数呈近似线性关系。溶剂和计算研究支持根据局域极性对伸缩频率的经验解释。为了开始探索利用这一技术来研究酶的功能和机制，我们报告了对CLHB还原酶中(甲基-d(3))蛋氨酸残基的初步分析。具体地说，我们表征了在催化重要的Met20环中的Met16和Met20以及位于酶疏水核心内的Met42的红外吸收。这些结果证实了二氢叶酸还原酶对其局部蛋白质环境的敏感性，证明了二氢叶酸还原酶在静电和动力学上的异质性，为直接表征蛋白质的静电学和动力学以及它们对催化的潜在贡献奠定了基础。
Much effort has been directed toward understanding the contributions of electrostatics and dynamics to protein function and especially to enzyme catalysis. Unfortunately, these studies have been limited by the absence of direct experimental probes, We have been developing the use of carbon-deuterium bonds as probes of proteins and now report the application of the technique to the enzyme clihydrofolate reductase, which catalyzes a hydride transfer and has served as a paradigm for biological catalysis. We observe that the stretching absorption frequency of (methyl-d(3)) methionine carbon-deuterium bonds shows an approximately linear dependence on solvent dielectric. Solvent and computational studies support the empirical interpretation of the stretching frequency in terms of local polarity. To begin to explore the use of this technique to study enzyme function and mechanism, we report a preliminary analysis of (methyl-d(3)) methionine residues within clihydrofolate reductase. Specifically, we characterize the IR absorptions at Met16 and Met20, within the catalytically important Met20 loop, and Met42, which is located within the hydrophobic core of the enzyme. The results confirm the sensitivity of the carbon-deuterium bonds to their local protein environment, demonstrate that dihydrofolate reductase is electrostatically and dynamically heterogeneous, and lay the foundation for the direct characterization protein electrostatics and dynamics and, potentially, their contribution to catalysis.