Journal
ACS CATALYSIS
Volume 10, Issue 14, Pages 7907-7914Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.0c01856
Keywords
dihydrofolate reductase; vibrational Stark effect; protein FTIR; protein dynamics; protein electrostatic; ligand-ligand interaction
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Funding
- MINECO [IJCI-201627503]
- Cardiff University
- Biotechnology and Biological Sciences Research Council (BBSRC) [BB/J005266, BB/L020394]
- Spanish Ministerio de Ciencia, Innovacion y Universidades [PGC2018-094852-B-C21]
- Generalitat Valenciana [AICO/2019/195]
- Universitat Jaume I [UJI~B2017-31]
- BBSRC [BB/L020394/1, BB/E008380/1] Funding Source: UKRI
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The contribution of ligand-ligand electrostatic interaction to transition state formation during enzyme catalysis has remained unexplored, even though electrostatic forces are known to play a major role in protein functions and have been investigated by the vibrational Stark effect (VSE). To monitor electrostatic changes along important steps during catalysis, we used a nitrile probe (T46C-CN) inserted proximal to the reaction center of three dihydrofolate reductases (DHFRs) with different biophysical properties, Escherichia coli DHFR (EcDHFR), its conformationally impaired variant (EcDHFR-S148P), and Geo bacillus stearothermophilus DHFR (BsDHFR). Our combined experimental and computational approach revealed that the electric field projected by the substrate toward the probe negates those exerted by the cofactor when both are bound within the enzymes. This indicates that compared to previous models that focus exclusively on subdomain reorganization and protein-ligand contacts, ligand-ligand interactions are the key driving force to generate electrostatic environments conducive for catalysis.
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