Illuminating the Mechanistic Roles of Enzyme Conformational Dynamics
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.
- Research Organization:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 1001563
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, 104(46):18055-18060, Journal Name: Proceedings of the National Academy of Sciences of the United States of America, 104(46):18055-18060 Journal Issue: 46 Vol. 104; ISSN PNASA6; ISSN 0027-8424
- Country of Publication:
- United States
- Language:
- English
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