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.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Gaweska H,Henderson Pozzi M,Schmidt DM,McCafferty DG,Fitzpatrick PF

doi

10.1021/bi900499w

subject

Has Abstract

pub_date

2009-06-16 00:00:00

pages

5440-5

issue

23

eissn

0006-2960

issn

1520-4995

journal_volume

48

pub_type

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