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

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

Research output: Contribution to journalArticlepeer-review

39 Scopus citations


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
Issue number7
StatePublished - Feb 20 2013

ASJC Scopus subject areas

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


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