title: Use of pH and kinetic isotope effects to establish chemistry as rate-limiting in oxidation of a peptide substrate by LSD1.
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, 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.
authors.affiliation: Department of Biochemistry and Biophysics and Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Helena
authors.initials: H
authors.lastname: Gaweska
authors.forename: Michelle
authors.initials: M
authors.lastname: Henderson Pozzi
authors.forename: Dawn M Z
authors.initials: DM
authors.lastname: Schmidt
authors.forename: Dewey G
authors.initials: DG
authors.lastname: McCafferty
authors.forename: Paul F
authors.initials: PF
authors.lastname: Fitzpatrick
mesh_terms: D001665:Binding Sites; D003903:Deuterium / Q000737:chemistry; D056466:Histone Demethylases; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007554:Isotopes / Q000737:chemistry; D007700:Kinetics; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D007554:Isotopes; D010455:Peptides; D003903:Deuterium; D056466:Histone Demethylases; C495195:KDM1A protein, human; D010089:Oxidoreductases, N-Demethylating