Hydrogen-Deuterium Exchange within Adenosine Deaminase, a TIM Barrel Hydrolase, Identifies Networks for Thermal Activation of Catalysis.
Hydrogen-Deuterium Exchange within Adenosine Deaminase, a TIM Barrel Hydrolase, Identifies Networks for Thermal Activation of Catalysis.
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DOI：
10.1021/jacs.0c07866
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发表时间：
2020-11-25
影响因子：
15
通讯作者：
Klinman JP
中科院分区：
文献类型：
--
作者：
Gao S;Thompson EJ;Barrow SL;Zhang W;Iavarone AT;Klinman JP
蛋白质本质上是柔性大分子，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的优化有关。时间、温度和突变依赖性氢氘交换与质谱法 (HDX-MS) 先前已被用来识别空间分辨的、与催化相关的酶动态区域。现在，我们扩展了这项技术，以探究多种广泛分布的 TIM 桶蛋白中蛋白质灵活性和化学反应性的相关性，以鼠腺苷脱氨酶 (mADA) 为靶点，该酶催化腺苷不可逆脱氨为肌苷和氨。对位于结合底物附近的第二球体苯丙氨酸的一系列突变进行速率和活化能的结构-功能分析后，选择具有较高活化能（Ea = 17.3 kcal/mol）的催化受损的 Phe61Ala 和野生型（WT）mADA（Ea = 11.5 kcal/mol）进行 HDX-MS 实验。肽段 HDX 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 蛋白质序列在 HDX 行为的温度依赖性方面表现出显着变化，其中主要变化是蛋白质灵活性的增加。利用 Phe61Ile（它显示与 WT 相同的 kcat 活化能）作为对照，我们能够进一步细化 HDX 分析，突出显示以功能相关方式改变的 mADA 区域。构建了一个图谱，说明了蛋白质区域，这些蛋白质区域对于活性位点配置的热优化至关重要，这些活性位点配置主导天然酶中的反应势垒穿越。
Proteins are intrinsically flexible macromolecules that undergo internal motions with timescales 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 wide-spread TIM barrel proteins, targeting a 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 = 17.3 kcal/mol) and the wild type (WT) mADA (Ea = 11.5 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.
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Tim Barrel结构促进了蛋白质介导的代谢的早期演变。
影响因子：
3.9
作者：
Goldman AD;Beatty JT;Landweber LF
通讯作者：
Landweber LF
影响因子：
14.8
作者：
Huang PS;Feldmeier K;Parmeggiani F;Velasco DAF;Höcker B;Baker D
通讯作者：
Baker D
DOI：
10.1016/j.jasms.2006.06.006
发表时间：
2006-11-01
影响因子：
3.2
作者：
Englander, S. Walter
通讯作者：
Englander, S. Walter
DOI：
10.1126/science.1259802
发表时间：
2014-12-19
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Fried SD;Bagchi S;Boxer SG
通讯作者：
Boxer SG
DOI：
10.1073/pnas.212637899
发表时间：
2002-12-10
影响因子：
11.1
作者：
Fenimore, PW;Frauenfelder, H;Parak, FG
通讯作者：
Parak, FG