Observing lysozyme's closing and opening motions by high-resolution single-molecule enzymology.
Observing lysozyme's closing and opening motions by high-resolution single-molecule enzymology.
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通过高分辨率的单分子酶学观察溶菌酶的闭合和打开动作。
DOI：
10.1021/cb500750v
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发表时间：
2015-06-19
影响因子：
4
通讯作者：
Collins PG
中科院分区：
文献类型：
--
作者：
Akhterov MV;Choi Y;Olsen TJ;Sims PC;Iftikhar M;Gul OT;Corso BL;Weiss GA;Collins PG
单分子技术可以监测酶开放和闭合构象之间的转换动力学，但这种方法通常缺乏分辨率来观察潜在的转换途径或中间构象动力学。我们已经使用了1 MHz带宽的碳纳米管晶体管，以电子方式监测单分子的酶T4溶菌酶，因为它处理基板。2 μs的实验分辨率允许直接记录溶菌酶的打开和关闭转变。出乎意料的是，这两个动作平均需要37 µs。转变持续时间的分布也与酶的状态（催化或非生产）无关。平滑，连续的过渡的观察表明，一个协调一致的机制，糖苷水解与溶菌酶的两个域关闭后，在其活性位点的多糖底物。我们将这些平滑运动与非协调机制区分开来，在大约10%的溶菌酶打开和关闭中观察到，其中酶在中间，部分闭合构象中暂停额外的40至140 µs。在中间成形事件期间，观察到的速率限制步骤的数量增加到四个，与逐步箭头推动机制中所需的四个步骤一致。这种中间构象的形成再次独立于酶的状态。总之，结果表明溶菌酶作为布朗马达运行。在这个模型中，酶跟踪一个单一的途径关闭和酶开放的反向途径，无论其瞬时催化生产力。观察到的对称性，在酶的开放和关闭，因此表明，底物易位发生，而酶是关闭的。
Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed conformations, but such methods usually lack the resolution to observe the underlying transition pathway or intermediate conformational dynamics. We have used a 1 MHz-bandwidth carbon nanotube transistor to electronically monitor single molecules of the enzyme T4 lysozyme as it processes substrate. An experimental resolution of 2 µs allowed the direct recording of lysozyme’s opening and closing transitions. Unexpectedly, both motions required 37 µs on average. The distribution of transition durations was also independent of the enzyme’s state, either catalytic or non-productive. The observation of smooth, continuous transitions suggests a concerted mechanism for glycoside hydrolysis with lysozyme’s two domains closing upon the polysaccharide substrate in its active site. We distinguish these smooth motions from a non-concerted mechanism, observed in approximately 10% of lysozyme openings and closings, in which the enzyme pauses for an additional 40 to 140 µs in an intermediate, partially closed conformation. During intermediate forming events, the number of rate limiting steps observed increases to four, consistent with four steps required in the step-wise, arrow-pushing mechanism. The formation of such intermediate conformations was again independent of the enzyme’s state. Taken together, the results suggest lysozyme operates as a Brownian motor. In this model, the enzyme traces a single pathway for closing and the reverse pathway for enzyme opening, regardless of its instantaneous catalytic productivity. The observed symmetry in enzyme opening and closing thus suggests that substrate translocation occurs while the enzyme is closed.