Substrate specificity and kinetic mechanism of mammalian G9a histone H3 methyltransferase
Substrate specificity and kinetic mechanism of mammalian G9a histone H3 methyltransferase
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DOI：
10.1074/jbc.m409604200
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发表时间：
2004-12-17
影响因子：
4.8
通讯作者：
Pradhan, S
中科院分区：
文献类型：
--
作者：
Patnaik, D;Chin, HG;Pradhan, S
在杆状病毒表达系统中表达并纯化了赖氨酸特异性鼠组蛋白H3甲基转移酶G9 a。重组酶的一级结构与天然酶相同。酶活性在碱性条件（> pH 8）和低盐浓度下是有利的，并且在25和42 ℃之间几乎不变。纯化的G9 a用于底物特异性和稳态动力学分析，其中肽代表未二甲基化或二甲基化的赖氨酸9组蛋白H3尾，其具有天然赖氨酸4或赖氨酸4变为丙氨酸（K4 AK 9）。在体外，H3尾肽的甲基化导致Lys-9的三甲基化，并且反应是进行性的。在野生型和K4 AK 9组蛋白H3尾部，甲基化的转换数（k（cat））分别为88和32 h（-1）。野生型和K4 AK 9的米氏常数（K-m（pep））分别为0.9和1.0 μ M，S-腺苷-L-甲硫氨酸的米氏常数（K-m（pep））分别为1.8和0.6 μ M。重组组蛋白H3的动力学常数相当。K4 AK 9二-至三甲基-赖氨酸的转化比甲基基团添加至未甲基化肽慢7倍。预孵育研究表明，G9 a-Met和G9 a-肽复合物具有催化活性。用肽和S-腺苷-L-甲硫氨酸（SNAMet）的初始速度数据和用S-腺苷-L-同型半胱氨酸的产物抑制研究来评估反应的动力学机制。双倒数图和预孵育研究显示S-腺苷-L-同型半胱氨酸作为竞争性抑制剂对p53 Met和混合抑制剂对肽。三甲基化肽充当底物肽的竞争性抑制剂和BCMet的混合抑制剂，表明重组G9 a的Bi Bi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。
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 degreesC. 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 muM and for S-adenosyl-L-methionine (K-m(AdoMet)) were 1.8 and 0.6 muM, 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.