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

Aduragbemi S. Adesina, Louis Y. P. Luk, Rudolf K. Allemann

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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.

Original languageEnglish
Pages (from-to)2410-2414
Number of pages5
JournalChemBioChem
Volume22
Issue number14
Early online date19 Apr 2021
DOIs
Publication statusPublished - 15 Jul 2021

Keywords

  • cryo-kinetics
  • dihydrofolate reductase
  • protein dynamics
  • heavy enzyme
  • isotope effects
  • pre-steady state kinetics

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