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Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to poor experimental access. This shortcoming is evident withEscherichia colidihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we describe ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments to map the conformational dynamics of the Michaelis complex of DHFR. We resolve coupled global and local motions and find that these motions are engaged by the protonated substrate to promote efficient catalysis. This result suggests a fundamental design principle for multistep enzymes in which pre-existing dynamics enable intermediates to drive rapid electrostatic reorganization to facilitate subsequent chemical steps.
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This content will become publicly available on December 9, 2026
Substrate structure modulates the catalytic dynamics of HDAC8 at the single-molecule level
Understanding how substrate structure alters an enzyme's conformational landscape is central to catalyst design. Using single-molecule electronic sensors, we reveal how substitutions on an HDAC8 substrate modulate the enzyme's underlying catalytic dynamics. We demonstrate that a trifluoroacetyl group accelerates catalysis, while a Boc cap and an allosteric activator synergistically simplify the kinetic pathway by stabilizing productive conformations. These findings provide direct, real-time insight into how substrate-induced conformational dynamics control enzyme catalysis.
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- Award ID(s):
- 2317111
- PAR ID:
- 10654187
- Publisher / Repository:
- Royal Society of Chemistry
- Date Published:
- Journal Name:
- Catalysis Science & Technology
- Volume:
- 15
- Issue:
- 24
- ISSN:
- 2044-4753
- Page Range / eLocation ID:
- 7324-7328
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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