Catalytic properties and kinetic mechanism of human recombinant lys-9 histone H3 methyltransferase SUV39H1:: Participation of the chromodomain in enzymatic catalysis
Catalytic properties and kinetic mechanism of human recombinant lys-9 histone H3 methyltransferase SUV39H1:: Participation of the chromodomain in enzymatic catalysis
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DOI：
10.1021/bi051997r
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发表时间：
2006-03-14
期刊：
影响因子：
2.9
通讯作者：
Pradhan, S
中科院分区：
文献类型：
--
作者：
Chin, HG;Patnaik, D;Pradhan, S
H3赖氨酸9（H3K9）甲基化是基因调控和染色质组织的主要组成部分。SUV39H1在臂间异染色质区域甲基化H3K9，并参与维持基因组稳定性。在这项研究中，重组纯化的SUV 39 H1用于底物特异性和稳态动力学分析，肽代表未或二甲基化赖氨酸9组蛋白H3尾或全长人重组H3（rH3）。重组SUV 39 H1通过非进行性机制甲基化其底物。无论是肽或pH6Met首先结合到酶的催化能力的二元复合物。SUV 39 H1的产物抑制研究表明，S-腺苷-L-同型半胱氨酸是S-腺苷-L-蛋氨酸的竞争性抑制剂，也是底物肽的混合抑制剂。类似地，甲基化的肽是未甲基化的肽的竞争性抑制剂，并且是C3Met的混合抑制剂，这表明重组SUV 39 H1的双-双反应中的随机机制，其中任一底物可以首先与酶结合，并且任一产物可以首先释放。H3尾肽和rH3的转换数（k（cat））相当（分别为12和8 h（-1）），而相同的二甲基化赖氨酸9 H3尾肽的转换数为1.5 h（-1）。甲基化肽的米氏常数（K-m（pep））比未甲基化肽的米氏常数低13倍。对于未甲基化的肽底物和rH3，K-m（K-Met）的米氏常数分别为12和6 μ M。在高浓度的rH3下观察到甲基化水平的降低，这意味着底物抑制。缺失的chromodomain或点突变的保守氨基酸，W64A或W67A，SUV 39 H1的酶活性受损，尽管存在一个完整的催化SET域。因此，SUV 39 H1利用色结构域和SET结构域进行催化。
Historic 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 mu M 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.