Department of Chemistry, Emory University , Atlanta, Georgia 30322, United States.
Department of Chemistry, University of Washington , Seattle, Washington 98195, United States.
J Am Chem Soc. 2018 Jan 24;140(3):900-903. doi: 10.1021/jacs.7b12369. Epub 2018 Jan 11.
Previous studies of a thermophilic alcohol dehydrogenase (ht-ADH) demonstrated a range of discontinuous transitions at 30 °C that include catalysis, kinetic isotope effects, protein hydrogen-deuterium exchange rates, and intrinsic fluorescence properties. Using the Förster resonance energy transfer response from a Trp-NADH donor-acceptor pair in T-jump studies of ht-ADH, we now report microsecond protein motions that can be directly related to active site chemistry. Two distinctive transients are observed: a slow, kinetic process lacking a temperature break, together with a faster transient that is only detectable above 30 °C. The latter establishes a link between enzyme activity and microsecond protein motions near the cofactor binding site, in a region distinct from a previously detected protein network that communicates with the substrate binding site. Though evidence of direct dynamical links between microsecond protein motions and active site bond cleavage events is extremely rare, these studies highlight the potential of T-jump measurements to uncover such properties.
先前对嗜热醇脱氢酶（ht-ADH）的研究表明，在 30°C 下存在一系列不连续的转变，包括催化、动力学同位素效应、蛋白质氢氘交换速率和固有荧光性质。使用 T-跳跃研究中来自 Trp-NADH 供体-受体对的Förster 共振能量转移响应，我们现在报告了可以直接与活性位点化学相关的微秒蛋白质运动。观察到两个独特的瞬变：一个缓慢的、缺乏温度中断的动力学过程，以及一个仅在 30°C 以上才能检测到的较快瞬变。后者在辅酶结合位点附近的区域建立了酶活性与微秒蛋白质运动之间的联系，该区域与先前检测到的与底物结合位点通信的蛋白质网络不同。尽管微秒蛋白质运动与活性位点键断裂事件之间存在直接动力学联系的证据极为罕见，但这些研究强调了 T-跳跃测量法揭示此类性质的潜力。
