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. 2009 Jun 16;48(23):5440-5.
doi: 10.1021/bi900499w.

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

Helena Gaweska  1 , Michelle Henderson Pozzi, Dawn M Z Schmidt, Dewey G McCafferty, Paul F Fitzpatrick
Affiliations

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

Helena Gaweska et al. Biochemistry. .

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.

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Figures

Scheme 1
Scheme 1
Figure 1
Figure 1
kcat/KM pH profiles for LSD1 with protiated (closed circles) and deuterated (open squares) H3K4 21-mer dimethylated peptide. The lines are from fits of the data to eq 1.
Figure 2
Figure 2
Spectral changes during reduction of LSD1 by the H3K4 21-mer dimethylated peptide: A, time course at 458 nm with protiated (closed circles) or deuterated (open circles) substrate (only 1/30th of the points are shown for clarity); B, Changes in the entire visible absorbance spectrum during reduction by the deuterated substrate; C, Initial (−) and final (⋯) spectra upon LSD1 flavin reduction by deuterated H3K4 21-mer dimethyl peptide obtained from a global analysis of the data in B using a single-step kinetic model. The lines in A are from fits of the data to eq 3.
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