Department of Physics, Emory University, Atlanta, Georgia.
Department of Physics, Emory University, Atlanta, Georgia.
Biophys J. 2018 Jun 19;114(12):2775-2786. doi: 10.1016/j.bpj.2018.03.039.
The adenosylcobalamin- (coenzyme B) dependent ethanolamine ammonia-lyase (EAL) plays a key role in aminoethanol metabolism, associated with microbiome homeostasis and Salmonella- and Escherichia coli-induced disease conditions in the human gut. To gain molecular insight into these processes toward development of potential therapeutic targets, reactions of the cryotrapped (S)-2-aminopropanol substrate radical EAL from Salmonella typhimurium are addressed over a temperature (T) range of 220-250 K by using T-step reaction initiation and time-resolved, full-spectrum electron paramagnetic resonance spectroscopy. The observed substrate radical reaction kinetics are characterized by two pairs of biexponential processes: native decay to diamagnetic products and growth of a non-native radical species and Co(II) in cobalamin. The multicomponent low-T kinetics are simulated by using a minimal model, in which the substrate-radical macrostate, S, is partitioned by a free-energy barrier into two sequential microstates: 1) S, a relatively high-entropy/high-enthalpy microstate with a protein configuration that captures the nascent substrate radical in the terminal step of radical-pair separation; and 2) S, a relatively low-enthalpy/low-entropy microstate with a protein configuration that enables the rearrangement reaction. The non-native, destructive reaction of S at T ≤ 250 K is caused by a prolonged lifetime in the substrate-radical capture state. Monotonic S decay over 278-300 K indicates that the free-energy barrier to S and S interconversion is latent at physiological T-values. Overall, the low-temperature studies reveal two protein-configuration microstates and connecting protein-configurational transitions that specialize the S macrostate for the dual functional roles of radical capture and rearrangement enabling. The identification of new, to our knowledge, intermediate states and specific protein-fluctuation contributions to the reaction coordinate represent an advance toward development of novel therapeutic targets in EAL.
腺钴胺素（辅酶 B）依赖的乙醇胺氨裂解酶（EAL）在氨基酸乙醇代谢中起着关键作用，与微生物组的动态平衡以及沙门氏菌和大肠杆菌在人类肠道中引起的疾病状态有关。为了深入了解这些过程，我们针对来自鼠伤寒沙门氏菌的冷冻捕获（S）-2-氨基-1-丙醇底物自由基 EAL，在 220-250 K 的温度范围内进行了研究，采用 T 步反应引发和时间分辨、全谱电子顺磁共振波谱。观察到的底物自由基反应动力学特征是两对双指数过程：原生衰减为抗磁性产物和非原生自由基物种以及钴胺素中的 Co(II)的生长。使用最小模型模拟多组分低温动力学，其中将底物-自由基宏观状态 S 通过自由能势垒分为两个连续的微状态：1）S，一个相对高熵/高焓的微状态，具有捕获在自由基对分离的最后一步中新生的底物自由基的蛋白质构象；2）S，一个相对低焓/低熵的微状态，具有能够进行重排反应的蛋白质构象。在 T≤250 K 时，S 的非原生、破坏性反应是由于在底物-自由基捕获状态下的寿命延长引起的。在 278-300 K 范围内 S 的单调衰减表明，S 和 S 相互转化的自由能势垒在生理 T 值下是潜伏的。总体而言，低温研究揭示了两种蛋白质构象微状态和连接蛋白质构象的转变，这些转变使 S 宏观状态专门用于自由基捕获和重排的双重功能角色。鉴定新的、据我们所知的中间状态和特定蛋白质波动对反应坐标的贡献，代表了在 EAL 中开发新型治疗靶点的进展。
