Illuminating the mechanistic roles of enzyme conformational dynamics

*Department of Chemistry,
^†Biophysics Graduate Group, and
^¶Department of Chemical Engineering, University of California, Berkeley, CA 94720; and
^‖Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720

Communicated by Judith P. Klinman, University of California, Berkeley, CA, September 12, 2007 (received for review July 2, 2007)

Abstract

Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme's lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK's domain movements on its catalytic time scale. To quantitatively measure the enzyme's entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly, the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme's conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme's rate-limiting step.

Footnotes

**To whom correspondence should be addressed. E-mail: hawyang{at}berkeley.edu
Author contributions: J.A.H., K.D., L.P.W., S.B., and H.Y. designed research; J.A.H., K.D., and S.B. performed research; J.B. and J.-W.C. contributed new reagents/analytic tools; J.A.H., K.D., and H.Y. analyzed data; and J.A.H. and H.Y. wrote the paper.
↵ ^‡Present address: Harvard Law School, Cambridge, MA 02138.
↵ ^§Present address: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0708600104/DC1.
Abbreviations:

AK,

adenylate kinase;

AMP-PNP,

adenosine 5′-[β,γ-imido]triphosphate.