O'Rourke Kathleen F, Axe Jennifer M, D'Amico Rebecca N, Sahu Debashish, Boehr David D
Department of Chemistry, The Pennsylvania State University, University Park, PA, United States.
Front Mol Biosci. 2018 Nov 8;5:92. doi: 10.3389/fmolb.2018.00092. eCollection 2018.
Tryptophan synthase is a model system for understanding allosteric regulation within enzyme complexes. Amino acid interaction networks were previously delineated in the isolated alpha subunit (αTS) in the absence of the beta subunit (βTS). The amino acid interaction networks were different between the ligand-free enzyme and the enzyme actively catalyzing turnover. Previous X-ray crystallography studies indicated only minor localized changes when ligands bind αTS, and so, structural changes alone could not explain the changes to the amino acid interaction networks. We hypothesized that the network changes could instead be related to changes in conformational dynamics. As such, we conducted nuclear magnetic resonance relaxation studies on different substrate- and products-bound complexes of αTS. Specifically, we collected N R relaxation dispersion data that reports on microsecond-to-millisecond timescale motion of backbone amide groups. These experiments indicated that there are conformational exchange events throughout αTS. Substrate and product binding change specific motional pathways throughout the enzyme, and these pathways connect the previously identified network residues. These pathways reach the αTS/βTS binding interface, suggesting that the identified dynamic networks may also be important for communication with the βTS subunit.
色氨酸合酶是理解酶复合物中变构调节的一个模型系统。之前在没有β亚基（βTS）的情况下，已在分离出的α亚基（αTS）中描绘了氨基酸相互作用网络。无配体酶和积极催化周转的酶之间的氨基酸相互作用网络是不同的。先前的X射线晶体学研究表明，当配体结合αTS时，只有微小的局部变化，因此，仅结构变化无法解释氨基酸相互作用网络的变化。我们推测，网络变化可能反而与构象动力学的变化有关。因此，我们对αTS的不同底物结合和产物结合复合物进行了核磁共振弛豫研究。具体而言，我们收集了N R弛豫色散数据，该数据报告了主链酰胺基团在微秒到毫秒时间尺度上的运动。这些实验表明，整个αTS中都存在构象交换事件。底物和产物结合改变了整个酶的特定运动途径，并且这些途径连接了先前确定的网络残基。这些途径延伸至αTS/βTS结合界面，表明所确定的动态网络可能对于与βTS亚基的通信也很重要。
