Huang Y T, Liaw Y C, Gorbatyuk V Y, Huang T H
Institute of Biomedical Sciences, Nankang Taipei, Taiwan, 11529, Rupublic of China.
J Mol Biol. 2001 Apr 6;307(4):1075-90. doi: 10.1006/jmbi.2001.4539.
Escherichia coli thioesterase/protease I (TEP-I) is a member of a novel subclass of the lipolytic enzymes with a distinctive GDSLS motif. In addition to possessing thioesterase and protease activities, TEP-I also exhibits arylesterase activity. We have determined the (15)N nuclear magnetic spin relaxation rates, R(1) and R(2), and the steady state (1)H-(15)N heteronuclear Overhauser effect, measured at both 11.74 T and 14.09 T, of (u-(15)N) TEP-I. These data were analyzed using model-free formalism (with axially symmetric rotational diffusion anisotropy) to extract the backbone dynamics of TEP-I. The results reveal that the core structure of the central beta-sheet and the long alpha-helices are rigid, while the binding pocket appears to be rather flexible. The rigid core serves as a scaffold to anchor the essential loops, which form the binding pocket. The most flexible residues display large amplitude fast (ps/ns time-scale) motion and lie on one stripe whose orientation is presumed to be the ligand-binding orientation. We also detected the presence of several residues displaying slow (microseconds/ms time-scale) conformational exchanging processes. These residues lie around the binding pocket and are oriented perpendicularly to the orientation of the flexible stripe. Two of the putative catalytic triads, Ser10 and His157, and their neighbors show motion on the microseconds/ms time-scale, suggesting that their slow motion may have a role in catalysis, in addition to their possible roles in ligand binding. The presence of a flexible substrate-binding pocket may also facilitate binding to a wide range of substrates and confer the versatile functional property of this protein.
大肠杆菌硫酯酶/蛋白酶I（TEP-I）是具有独特GDSLS基序的新型脂解酶亚类的成员。除了具有硫酯酶和蛋白酶活性外，TEP-I还表现出芳基酯酶活性。我们已经测定了（μ-（15）N）TEP-I在11.74 T和14.09 T下的（15）N核磁共振弛豫率R(1)和R(2)，以及稳态（1）H-（15）N异核Overhauser效应。使用无模型形式主义（具有轴对称旋转扩散各向异性）对这些数据进行分析，以提取TEP-I的主链动力学。结果表明，中央β-折叠和长α-螺旋的核心结构是刚性的，而结合口袋似乎相当灵活。刚性核心作为支架来锚定形成结合口袋的关键环。最灵活的残基表现出大幅度快速（皮秒/纳秒时间尺度）运动，位于一条条纹上，其方向被认为是配体结合方向。我们还检测到几个残基存在缓慢（微秒/毫秒时间尺度）的构象交换过程。这些残基位于结合口袋周围，且垂直于灵活条纹的方向排列。两个推定的催化三联体Ser10和His157及其相邻残基在微秒/毫秒时间尺度上表现出运动，这表明它们的缓慢运动除了可能在配体结合中发挥作用外，还可能在催化中起作用。灵活的底物结合口袋的存在也可能有助于与多种底物结合，并赋予该蛋白质多功能特性。
