David R. Cheriton School of Computer Science, University of Waterloo, Waterloo, ON N2L 3G1, Canada.
J Am Chem Soc. 2020 Nov 25;142(47):19936-19949. doi: 10.1021/jacs.0c07866. Epub 2020 Nov 12.
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 (a = 7.5 kcal/mol) and the wild type (WT) mADA (a = 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 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.
蛋白质是具有内在柔韧性的大分子，其内部运动的时间尺度跨越飞秒到毫秒。这些波动与酶催化反应的反应势垒优化有关。时间、温度和突变依赖性氘氢交换与质谱（HDX-MS）结合以前曾被用来识别酶的空间分辨、催化相关动态区域。我们现在将这项技术扩展到探索 TIM 桶蛋白中广泛存在的蛋白质柔韧性和化学反应性之间的相关性，目标是靶向催化不可逆腺苷脱氨酶（mADA），该酶催化腺苷不可逆脱氨为肌苷和氨。在对一系列位于接近结合底物的第二球苯丙氨酸的突变进行速率和活化能的结构-功能分析之后，选择催化受损的 Phe61Ala 和野生型（WT）mADA 进行 HDX-MS 实验，它们的活化能分别升高（a = 7.5 kcal/mol）和（a = 5.0 kcal/mol）。肽段的 HDX 速率常数和活化能被定量，并用于评估突变依赖性的局部和远程运动变化。分析表明，Phe61Ala 的约 50%的蛋白质序列显示出 HDX 行为的温度依赖性的显著变化，主要变化是蛋白质柔韧性增加。利用 Phe61Ile 作为对照，其具有与 WT 相同的 ，我们能够进一步细化 HDX 分析，突出 mADA 的功能相关方式发生变化的区域。构建了一个图谱，说明了提议对热优化主导天然酶中反应势垒穿越的活性位点构象的蛋白质区域是必需的。
