The analysis of hydrogen deuterium exchange by mass spectrometry as a function of temperature and mutation (TDHDX-MS) has emerged as a generic and efficient tool for the spatial resolution of protein networks that are proposed to function in the thermal activation of catalysis. In this work, we extend TDHDX from apo-enzyme structures to protein-ligand complexes. Using adenosine deaminase as a prototype, we compared the impacts of a substrate analog (1-deaza-adenosine or DAA) and a very tight-binding inhibitor/transition state analog (pentostatin) at single and multiple temperatures. At a single temperature, we observed different HDX-MS properties for the two ligands, as expected from their 10⁶-fold differences in strength of binding. By contrast, analogous patterns for TDHDX-MS emerge in the presence of both DAA and pentostatin, indicating similar impacts of either ligand on the enthalpic barriers for local protein unfolding. We extended TDHDX to a function-altering mutant of adenosine deaminase in the presence of pentostatin and revealed a protein thermal network that is highly similar to that previously reported for the apo-enzyme (Gao et al., 2020, JACS 142, 19936-19949). Finally, we discuss the differential impacts of pentostatin binding on overall protein flexibility vs. site-specific thermal transfer pathways in the context of models for substrate-induced changes to a distributed protein conformational landscape that act in synergy with embedded protein thermal networks to achieve efficient catalysis.

通过质谱法分析氢氘交换作为温度和突变的函数 (TDHDX-MS) 已成为一种通用且有效的工具，用于空间分辨率蛋白质网络，这些蛋白质网络被提议在催化的热激活中发挥作用。在这项工作中，我们将 TDHDX 从脱辅基酶结构扩展到蛋白质-配体复合物。使用腺苷脱氨酶作为原型，我们比较了底物类似物（1-脱氮腺苷或 DAA）和结合非常紧密的抑制剂/过渡态类似物（喷司他丁）在单一和多个温度下的影响。在单一温度下，我们观察到两种配体的不同 HDX-MS 特性，正如它们的 10 ^{6所预期的那样}-结合强度的倍数差异。相比之下，TDHDX-MS 的类似模式出现在同时存在 DAA 和喷司他丁的情况下，表明这两种配体对局部蛋白质展开的焓屏障具有相似的影响。我们将 TDHDX 扩展到存在喷司他丁的腺苷脱氨酶的功能改变突变体，并揭示了一个与之前报道的载脂酶高度相似的蛋白质热网络（Gao et al., 2020, JACS 142, 19936-19949 ). 最后，我们在底物诱导的分布式蛋白质构象景观模型的背景下讨论了喷司他丁结合对整体蛋白质灵活性与位点特异性热传递途径的不同影响，这些构象景观与嵌入式蛋白质热网络协同作用以实现高效催化。