title: Catalytic properties and kinetic mechanism of human recombinant Lys-9 histone H3 methyltransferase SUV39H1: participation of the chromodomain in enzymatic catalysis.
abstract: Histone H3 lysine 9 (H3K9) methylation is a major component of gene regulation and chromatin organization. SUV39H1 methylates H3K9 at the pericentric heterochromatin region and participates in the maintenance of genome stability. In this study, a recombinant purified SUV39H1 is used for substrate specificity and steady-state kinetic analysis with peptides representing the un- or dimethylated lysine 9 histone H3 tail or full-length human recombinant H3 (rH3). Recombinant SUV39H1 methylated its substrate via a nonprocessive mechanism. Binding of either peptide or AdoMet first to the enzyme made a catalytically competent binary complex. Product inhibition studies with SUV39H1 showed that S-adenosyl-l-homocysteine is a competitive inhibitor of S-adenosyl-l-methionine and a mixed inhibitor of substrate peptide. Similarly, the methylated peptide was a competitive inhibitor of the unmethylated peptide and a mixed inhibitor of AdoMet, suggesting a random mechanism in a bi-bi reaction for recombinant SUV39H1 in which either substrate can bind to the enzyme first and either product can release first. The turnover numbers (k(cat)) for the H3 tail peptide and rH3 were comparable (12 and 8 h(-)(1), respectively) compared to the value of 1.5 h(-)(1) for an identical dimethylated lysine 9 H3 tail peptide. The Michaelis constant for the methylated peptide (K(m)(pep)) was 13-fold lower compared to that of the unmethylated peptide. The Michaelis constants for AdoMet (K(m)(AdoMet)) were 12 and 6 microM for the unmethylated peptide substrate and rH3, respectively. A reduction in the level of methylation was observed at high concentrations of rH3, implying substrate inhibition. Deletion of the chromodomain or point mutation of the conserved amino acids, W64A or W67A, of SUV39H1 impaired enzyme activity despite the presence of an intact catalytic SET domain. Thus, SUV39H1 utilizes both the chromodomain and the SET domain for catalysis.
authors.affiliation: New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA.
authors.forename: Hang Gyeong
authors.initials: HG
authors.lastname: Chin
authors.forename: Debasis
authors.initials: D
authors.lastname: Patnaik
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Estève
authors.forename: Steven E
authors.initials: SE
authors.lastname: Jacobsen
authors.forename: Sriharsa
authors.initials: S
authors.lastname: Pradhan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011496:Protein Methyltransferases; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012435:S-Adenosylhomocysteine / Q000378:metabolism / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000378:metabolism / Q000494:pharmacology; D018411:Spodoptera / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D002843:Chromatin; D003067:Coenzymes; D006657:Histones; D010455:Peptides; D011994:Recombinant Proteins; D012097:Repressor Proteins; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; C407843:SUV39H1 protein, human; D000076983:Histone Methyltransferases; D008780:Methyltransferases; D011496:Protein Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D008239:Lysine