cb500750v_si_001.pdf (1.08 MB)
Download fileObserving Lysozyme’s Closing and Opening Motions by High-Resolution Single-Molecule Enzymology
journal contribution
posted on 2015-06-19, 00:00 authored by Maxim
V. Akhterov, Yongki Choi, Tivoli J. Olsen, Patrick C. Sims, Mariam Iftikhar, O. Tolga Gul, Brad L. Corso, Gregory A. Weiss, Philip G. CollinsSingle-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 nonproductive. 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 nonconcerted mechanism,
observed in approximately 10% of lysozyme openings and closings, in
which the enzyme pauses for an additional 40–140 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 stepwise, 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.