Abstract
Lysine-specific murine histone H3 methyltransferase, G9a, was expressed and purified in a baculovirus expression system. The primary structure of the recombinant enzyme is identical to the native enzyme. Enzymatic activity was favorable at alkaline conditions (>pH 8) and low salt concentration and virtually unchanged between 25 and 42 degrees C. Purified G9a was used for substrate specificity and steady-state kinetic analysis with peptides representing un- or dimethylated lysine 9 histone H3 tails with native lysine 4 or with lysine 4 changed to alanine (K4AK9). In vitro methylation of the H3 tail peptide resulted in trimethylation of Lys-9 and the reaction is processive. The turnover number (k(cat)) for methylation was 88 and 32 h(-1) on the wild type and K4AK9 histone H3 tail, respectively. The Michaelis constants for wild type and K4AK9 ((K(m)(pep))) were 0.9 and 1.0 microM and for S-adenosyl-L-methionine (K(m)(AdoMet)) were 1.8 and 0.6 microM, respectively. Comparable kinetic constants were obtained for recombinant histone H3. The conversion of K4AK9 di- to trimethyl-lysine was 7-fold slower than methyl group addition to unmethylated peptide. Preincubation studies showed that G9a-AdoMet and G9a-peptide complexes are catalytically active. Initial velocity data with peptide and S-adenosyl-L-methionine (AdoMet) and product inhibition studies with S-adenosyl-L-homocysteine were performed to assess the kinetic mechanism of the reaction. Double reciprocal plots and preincubation studies revealed S-adenosyl-L-homocysteine as a competitive inhibitor to AdoMet and mixed inhibitor to peptide. Trimethylated peptides acted as a competitive inhibitor to substrate peptide and mixed inhibitor to AdoMet suggesting a random mechanism in a Bi Bi reaction for recombinant G9a where either substrate can bind first to the enzyme, and either product can release first.
Highlights
Histones participate in packaging of eukaryotic DNA
Purified G9a was used for substrate specificity and steady-state kinetic analysis with peptides representing un- or dimethylated lysine 9 histone H3 tails with native lysine 4 or with lysine 4 changed to alanine (K4AK9)
Loss of G9a resulted in reduction of H3-K9 methylation in the Prader-Willi syndrome imprinting center resulting in disruption of CpG methylation of the Prader-Willi syndrome imprinting center in mouse ES cells [21]
Summary
Histone H3 Methyltransferase G9a Transfer Vector—G9a expression constructs were derived from pZKmG9aL, accession number AB077210 A typical reaction contained S-adenosyl-L-[methyl3H]methionine (AdoMet) (specific activity 15 Ci/mmol, Amersham Biosciences), substrate peptide, and enzyme in assay buffer (50 mM Tris-HCl, pH 9.0, 5 mM MgCl2, 4 mM dithiothreitol, 7 g/ml phenylmethylsulfonyl fluoride). To determine the sequence specificity of G9a methylation, 740 nM G9afl enzyme with 192 M synthetic peptide substrate (K4AK9) and 1.0 mM AdoMet was added in a total reaction volume of 300 l. Processivity Studies—A master mixture (600 l) containing 1.1 M biotin-conjugated histone H3 tail peptide (Wt-H3), 25 nM G9a, 5 M AdoMet was incubated at room temperature for 3 min. The reaction was started by adding the missing reactant, i.e. peptide (final concentration 4.4 M) in the first reaction mixture and S-adenosyl-L-[methyl-3H]methionine (5 M) to the second one.
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