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

Published

Journal Article

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, k(red), equals k(cat), 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 k(red), k(cat), and k(cat)/K(m) 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 k(cat)/K(m) 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)(k(cat)/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.

Full Text

Duke Authors

Cited Authors

  • Gaweska, H; Henderson Pozzi, M; Schmidt, DMZ; McCafferty, DG; Fitzpatrick, PF

Published Date

  • June 2009

Published In

Volume / Issue

  • 48 / 23

Start / End Page

  • 5440 - 5445

PubMed ID

  • 19408960

Pubmed Central ID

  • 19408960

Electronic International Standard Serial Number (EISSN)

  • 1520-4995

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi900499w

Language

  • eng