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Cryo‐kinetics Reveal Dynamic Effects on the Chemistry of Human Dihydrofolate Reductase
Author(s) -
Adesina Aduragbemi S.,
Luk Louis Y. P.,
Allemann Rudolf K.
Publication year - 2021
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202100017
Subject(s) - kinetics , dihydrofolate reductase , enzyme kinetics , chemistry , enzyme , kinetic isotope effect , reaction rate constant , catalysis , stereochemistry , enzyme catalysis , chemical kinetics , biochemistry , active site , deuterium , physics , quantum mechanics
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 ( k H LE / k H HE ) 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.