Illuminating the mechanistic roles of enzyme conformational dynamics

Jeffrey A. Hanson, Karl Duderstadt, Lucas P. Watkins, Sucharita Bhattacharyya, Jason Brokaw, Jhih Wei Chu, Haw Yang

Research output: Contribution to journalArticle

202 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)18055-18060
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number46
DOIs
StatePublished - Nov 13 2007
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • Adenylate kinase
  • Conformational equilibrium
  • Rate-limiting step
  • Single-molecule FRET

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