Use of pH and Kinetic Isotope Effects To Establish Chemistry as Rate-Limiting in Oxidation of a Peptide Substrate by LSD1

• Published in: Biochemistry (Easton) 2009-06, Vol.48 (23), p.5440-5445
• Main Authors: Gaweska, Helena, Henderson Pozzi, Michelle, Schmidt, Dawn M. Z, McCafferty, Dewey G, Fitzpatrick, Paul F
• Format: Article
• Language: English
• Subjects: Binding Sites; Deuterium - chemistry; Histone Demethylases; Humans; Hydrogen-Ion Concentration; Isotopes - chemistry; Kinetics; Oxidation-Reduction; Oxidoreductases, N-Demethylating - chemistry; Oxidoreductases, N-Demethylating - metabolism; Peptides - chemistry; Peptides - metabolism; Structure-Activity Relationship; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi900499w

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.