Abstract
Here we investigated the role of protein isotope labeling as a powerful technique to probe functionally important motions in enzyme catalysis and to study the conformational dynamics of proteins. We focused on dihydrofolate reductase (DHFR) and compared enzymes from organisms adapted to different temperature ranges. Previous studies indicated that dynamic coupling is detrimental to catalysis by DHFR from the mesophile Escherichia coli (EcDHFR). Our analysis suggested that dynamic coupling in DHFR catalysis, which arises from reorganizational motions necessary for facilitating charge transfer events, has been minimized during evolution. Contrary to the behavior observed for DHFR from the moderate thermophile Geobacillus stearothermophilus (BsDHFR), we found that the chemical transformation catalyzed by DHFR from the cold-adapted bacterium Moritella profunda (MpDHFR) is only weakly affected by protein isotope substitutions at low temperatures. However, the isotopically substituted enzyme becomes a significantly inferior catalyst at higher, non-physiological temperatures. Using QM/MM studies, we demonstrated that this behavior is caused by the enzyme’s structural sensitivity to temperature changes, which enhances unfavorable dynamic coupling at higher temperatures by promoting additional recrossing trajectories on the transition state dividing surface. We proposed that these motions are minimized through the fine-tuning of DHFR flexibility, optimizing the free energy surface of the reaction to maintain a nearly static reaction-ready configuration with optimal electrostatic properties under physiological conditions.
