Proteins are intrinsically flexible macromolecules that undergo internal motions with time scales spanning femtoseconds to milliseconds. These fluctuations are implicated in the optimization of reaction barriers for enzyme catalyzed reactions. Time, temperature, and mutation dependent hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has been previously employed to identify spatially resolved, catalysis-linked dynamical regions of enzymes. We now extend this technique to pursue the correlation of protein flexibility and chemical reactivity within the diverse and widespread TIM barrel proteins, targeting murine adenosine deaminase (mADA) that catalyzes the irreversible deamination of adenosine to inosine and ammonia. Following a structure-function analysis of rate and activation energy for a series of mutations at a second sphere phenylalanine positioned in proximity to the bound substrate, the catalytically impaired Phe61Ala with an elevated activation energy (Ea = 7.5 kcal/mol) and the wild type (WT) mADA (Ea = 5.0 kcal/mol) were selected for HDX-MS experiments. The rate constants and activation energies of HDX for peptide segments are quantified and used to assess mutation-dependent changes in local and distal motions. Analyses reveal that approximately 50% of the protein sequence of Phe61Ala displays significant changes in the temperature dependence of HDX behaviors, with the dominant change being an increase in protein flexibility. Utilizing Phe61Ile, which displays the same activation energy for kcat as WT, as a control, we were able to further refine the HDX analysis, highlighting the regions of mADA that are altered in a functionally relevant manner. A map is constructed that illustrates the regions of protein that are proposed to be essential for the thermal optimization of active site configurations that dominate reaction barrier crossings in the native enzyme.

中文翻译：

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腺苷脱氨酶（一种 TIM 桶水解酶）内的氢-氘交换可识别催化热活化网络

蛋白质本质上是灵活的大分子，其内部运动的时间跨度从飞秒到毫秒。这些波动与酶催化反应的反应屏障的优化有关。时间、温度和突变相关的氢-氘交换与质谱联用 (HDX-MS) 先前已被用于识别酶的空间解析、催化连接动态区域。我们现在扩展这项技术，以追求多样化和广泛的 TIM 桶蛋白中蛋白质灵活性和化学反应性的相关性，靶向催化腺苷不可逆脱氨为肌苷和氨的鼠腺苷脱氨酶 (mADA)。在对位于结合底物附近的第二个球体苯丙氨酸处的一系列突变的速率和活化能进行结构功能分析后，催化受损的 Phe61Ala 具有升高的活化能 (Ea = 7.5 kcal/mol) 和野生型(WT) mADA (Ea = 5.0 kcal/mol) 被选择用于 HDX-MS 实验。HDX 肽段的速率常数和活化能被量化并用于评估局部和远端运动的突变依赖性变化。分析表明，大约 50% 的 Phe61Ala 蛋白质序列显示出 HDX 行为的温度依赖性的显着变化，主要变化是蛋白质灵活性的增加。使用 Phe61Ile，它对 kcat 显示与 WT 相同的活化能，作为对照，我们能够进一步完善 HDX 分析，突出显示以功能相关方式改变的 mADA 区域。构建了一张图，说明了蛋白质的区域，这些区域被认为对于在天然酶中占主导地位的反应屏障交叉的活性位点配置的热优化是必不可少的。