Perturbative diffraction methods resolve a conformational switch that facilitates a two-step enzymatic mechanism

Greisman, Jack B.; Dalton, Kevin M.; Brookner, Dennis E.; Klureza, Margaret A.; Sheehan, Candice J.; Kim, In-Sik; Henning, Robert W.; Russi, Silvia; Hekstra, Doeke R.

doi:10.1073/pnas.2313192121

Title: Perturbative diffraction methods resolve a conformational switch that facilitates a two-step enzymatic mechanism

Journal Article · 22 February 2024 · Proceedings of the National Academy of Sciences of the United States of America

DOI: https://doi.org/10.1073/pnas.2313192121 · OSTI ID:2403417

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Harvard Univ., Cambridge, MA (United States); SLAC
Harvard Univ., Cambridge, MA (United States)
Argonne National Laboratory (ANL), Argonne, IL (United States); Univ. of Chicago, IL (United States). BioCARS
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)

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 with Escherichia coli dihydrofolate 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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Research Organization:: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States); Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institute of General Medical Sciences (NIGMS)

Grant/Contract Number:: AC02-76SF00515; AC02-06CH11357

OSTI ID:: 2403417

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 9 Vol. 121; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

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