Use of pH and kinetic isotope effects to establish chemistry as rate-limiting in oxidation of a peptide substrate by LSD1

Helena Gaweska, Michelle Henderson Pozzi, Dawn M.Z. Schmidt, Dewey G. McCafferty, Paul F. Fitzpatrick

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

The mechanism of oxidation of a peptide substrate by the flavoprotein lysine-specific demethylase (LSD1) has been examined using the effects of pH and isotopic substitution on steady-state and rapid-reaction kinetic parameters. The substrate contained the 21 N-terminal residues of histone H3, with a dimethylated lysyl residue at position 4. At pH 7.5, the rate constant for flavin reduction, kred, equals kcat, establishing the reductive half-reaction as rate-limiting at physiological pH. Deuteration of the lysyl methyls results in identical kinetic isotope effects of 3.1 ± 0.2 on the kred, kcat, and kcat/Km values for the peptide, establishing C-H bond cleavage as rate-limiting with this substrate. No intermediates between oxidized and reduced flavin can be detected by stopped-flow spectroscopy, consistent with the expectation for a direct hydride transfer mechanism. The kcat/Km value for the peptide is bell-shaped, consistent with a requirement that the nitrogen at the site of oxidation be uncharged and that at least one of the other lysyl residues be charged for catalysis. The D(kcat/K m) value for the peptide is pH-independent, suggesting that the observed value is the intrinsic deuterium kinetic isotope effect for oxidation of this substrate.

Original languageEnglish (US)
Pages (from-to)5440-5445
Number of pages6
JournalBiochemistry
Volume48
Issue number23
DOIs
StatePublished - Jun 16 2009

ASJC Scopus subject areas

  • Biochemistry

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