Capturing the Catalytic Proton of Dihydrofolate Reductase: Implications for General Acid–Base Catalysis

Wan, Qun; Bennett, Brad C.; Wymore, Troy; Li, Zhihong; Wilson, Mark A.; Brooks, III, Charles L.; Langan, Paul; Kovalevsky, Andrey; Dealwis, Chris G.

doi:10.1021/acscatal.1c00417

Title: Capturing the Catalytic Proton of Dihydrofolate Reductase: Implications for General Acid–Base Catalysis

Journal Article · Wed Apr 28 00:00:00 EDT 2021 · ACS Catalysis

DOI:https://doi.org/10.1021/acscatal.1c00417· OSTI ID:1779969

;

^[1]; Wymore, Troy ^[2]; Li, Zhihong;

^[3];

^[2]; Langan, Paul ^[4];

^[4]; Dealwis, Chris G.

Biological and Environmental Science Department, Samford University, Birmingham, Alabama 35229, United States
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588, United States
Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830, United States

Acid–base catalysis, which involves one or more proton transfer reactions, is a chemical mechanism commonly employed by many enzymes. The molecular basis for catalysis is often derived from structures determined at the optimal pH for enzyme activity. However, direct observation of protons from experimental structures is quite difficult; thus, a complete mechanistic description for most enzymes remains lacking. Dihydrofolate reductase (DHFR) exemplifies general acid–base catalysis, requiring hydride transfer and protonation of its substrate, DHF, to form the product, tetrahydrofolate (THF). Previous X-ray and neutron crystal structures coupled with theoretical calculations have proposed that solvent mediates the protonation step. However, visualization of a proton transfer has been elusive. Based on a 2.1 Å resolution neutron structure of a pseudo-Michaelis complex of E. coli DHFR determined at acidic pH, we report the direct observation of the catalytic proton and its parent solvent molecule. Comparison of X-ray and neutron structures elucidated at acidic and neutral pH reveals dampened dynamics at acidic pH, even for the regulatory Met20 loop. Guided by the structures and calculations, we propose a mechanism where dynamics are crucial for solvent entry and protonation of substrate. This mechanism invokes the release of a sole proton from a hydronium (H₃O⁺) ion, its pathway through a narrow channel that sterically hinders the passage of water, and the ultimate protonation of DHF at the N5 atom.

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Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

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

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1779969

Alternate ID(s):: OSTI ID: 1784106

Journal Information:: ACS Catalysis, Journal Name: ACS Catalysis Vol. 11 Journal Issue: 9; ISSN 2155-5435

Publisher:: American Chemical SocietyCopyright Statement

Country of Publication:: United States

Language:: English

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