Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis. Two isotopic hybrids were prepared; one with the mobile N‐terminal segment containing heavy isotopes (2H, 13C, 15N) and the remainder of the protein with natural isotopic abundance, and the other one with only the C‐terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N‐terminal segment affected only a physical step of catalysis, whereas the enzyme chemistry was affected by protein motions from the C‐terminal segment. QM/MM studies support the idea that dynamic effects on catalysis mostly originate from the C‐terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling, which is difficult to obtain with other studies, and helps define the dynamic networks of intramolecular interactions central to enzyme catalysis.

Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis.T wo isotopic hybrids were prepared;o ne with the mobile N-terminal segment containing heavy isotopes ( 2 H, 13 C, 15 N) and the remainder of the protein with natural isotopic abundance,and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only aphysical step of catalysis,whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/ MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling,w hichi sd ifficult to obtain with other studies,a nd helps define the dynamic networks of intramolecular interactions central to enzyme catalysis.

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 Geobacillus 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.

Effects of isotopic substitution on the rate constants of human dihydrofolate reductase (HsDHFR), an important target for anti‐cancer drugs, have not previously been characterized due to its complex fast kinetics. Here, we report the results of cryo‐measurements of the kinetics of the HsDHFR catalyzed reaction and the effects of protein motion on catalysis. Isotopic enzyme labeling revealed an enzyme KIE (kHLE/kHHE) close to unity above 0 °C; however, the enzyme KIE was increased to 1.72±0.15 at −20 °C, indicating that the coupling of protein motions to the chemical step is minimized under optimal conditions but enhanced at non‐physiological temperatures. The presented cryogenic approach provides an opportunity to probe the kinetics of mammalian DHFRs, thereby laying the foundation for characterizing their transition state structure.

Isotopologues, enzymes that differ only in their isotopic substitution, engage in a tug of war to determine the role of protein motions and electrostatics for catalysis. The features of the ribbon diagrams indicate that the enzymes are human dihydrofolate reductases. More information can be found in the Communication by R. K. Allemann et al.