Heavy-enzyme kinetic isotope effects on proton transfer in alanine racemase

Michael D. Toney, Joan Nieto Castro, Trevor A. Addington

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

The catalytic effects of perdeuterating the pyridoxal phosphate-dependent enzyme alanine racemase from Geobacillus stearothermophilus are reported. The mass of the heavy perdeuterated form is ∼5.5% greater than that of the protiated form, causing kinetic isotope effects (KIEs) of ∼1.3 on k cat and kcat/KM for both l-and d-alanine. These values increase when Cα-deuterated alanine is used as the substrate. The heavy-enzyme KIEs of ∼3 on kcat/KM with deuterated substrates are greater than the product of the individual heavy-enzyme and primary substrate KIEs. This breakdown of the rule of the geometric mean is likely due to coupled motion between the protein and the proton-transfer reaction coordinate in the rate-limiting step. These data implicate a direct role for protein vibrational motions in barrier crossing for proton-transfer steps in alanine racemase.

Original languageEnglish (US)
Pages (from-to)2509-2511
Number of pages3
JournalJournal of the American Chemical Society
Volume135
Issue number7
DOIs
StatePublished - Feb 20 2013

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Alanine Racemase
Enzyme kinetics
Proton transfer
Isotopes
Protons
Alanine
Substrates
Enzymes
Proteins
Geobacillus stearothermophilus
Pyridoxal Phosphate
Phosphates
Cats

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Heavy-enzyme kinetic isotope effects on proton transfer in alanine racemase. / Toney, Michael D.; Castro, Joan Nieto; Addington, Trevor A.

In: Journal of the American Chemical Society, Vol. 135, No. 7, 20.02.2013, p. 2509-2511.

Research output: Contribution to journalArticle

Toney, Michael D. ; Castro, Joan Nieto ; Addington, Trevor A. / Heavy-enzyme kinetic isotope effects on proton transfer in alanine racemase. In: Journal of the American Chemical Society. 2013 ; Vol. 135, No. 7. pp. 2509-2511.
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