Observing Lysozyme鈥檚 Closing and Opening Motions by High-Resolution Single-Molecule Enzymology
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- 作者：Maxim V. Akhterov ; Yongki Choi ; Tivoli J. Olsen ; Patrick C. Sims ; Mariam Iftikhar ; O. Tolga Gul ; Brad L. Corso ; Gregory A. Weiss ; Philip G. Collins
- 刊名：ACS Chemical Biology
- 出版年：2015
- 出版时间：June 19, 2015
- 年：2015
- 卷：10
- 期：6
- 页码：1495-1501
- 全文大小：330K
- ISSN：1554-8937
文摘
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 渭s allowed the direct recording of lysozyme鈥檚 opening and closing transitions. Unexpectedly, both motions required 37 渭s, on average. The distribution of transition durations was also independent of the enzyme鈥檚 state: either catalytic or nonproductive. The observation of smooth, continuous transitions suggests a concerted mechanism for glycoside hydrolysis with lysozyme鈥檚 two domains closing upon the polysaccharide substrate in its active site. We distinguish these smooth motions from a nonconcerted mechanism, observed in approximately 10% of lysozyme openings and closings, in which the enzyme pauses for an additional 40鈥?40 渭s 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 stepwise, arrow-pushing mechanism. The formation of such intermediate conformations was again independent of the enzyme鈥檚 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.