Substrate specificity and kinetic mechanism of mammalian G9a histone H3 methyltransferase.
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.
New England Biolabs, Beverly, MA 01915, USA.
Debasis
D
Patnaik
Hang Gyeong
HG
Chin
Pierre-Olivier
PO
Estève
Jack
J
Benner
Steven E
SE
Jacobsen
Sriharsa
S
Pradhan
D000409:Alanine / Q000737:chemistry; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001667:Binding, Competitive; D002384:Catalysis; D002460:Cell Line; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005822:Genetic Vectors; D016678:Genome; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry* / Q000378:metabolism; D006657:Histones / Q000737:chemistry; D006710:Homocysteine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008745:Methylation; D051379:Mice; D008956:Models, Chemical; D008957:Models, Genetic; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D020033:Protein Isoforms; D011496:Protein Methyltransferases; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D006657:Histones; D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D006710:Homocysteine; D000076983:Histone Methyltransferases; D011496:Protein Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D008239:Lysine; D000409:Alanine