Cryo-kinetics reveal dynamic effects on the chemistry of human dihydrofolate reductase

Adesina, Aduragbemi S., Luk, Louis Y. P. and Allemann, Rudolf K. (2021) Cryo-kinetics reveal dynamic effects on the chemistry of human dihydrofolate reductase. ChemBioChem, 22 (14). pp. 2410-2414. ISSN 1439-4227

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Abstract

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.

Item Type: Article
Additional Information: © 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.
Uncontrolled Keywords: cryo-kinetics,dihydrofolate reductase,protein dynamics,heavy enzyme,isotope effects,pre-steady state kinetics
Faculty \ School: Faculty of Science > School of Chemistry
Depositing User: LivePure Connector
Date Deposited: 24 Jun 2021 00:11
Last Modified: 21 Jul 2021 01:48
URI: https://ueaeprints.uea.ac.uk/id/eprint/80323
DOI: 10.1002/cbic.202100017

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