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