The mechanism of target base attack in DNA cytosine carbon 5 methylation
The mechanism of target base attack in DNA cytosine carbon 5 methylation
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DOI：
10.1021/bi0496743
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发表时间：
2004-09-14
期刊：
影响因子：
2.9
通讯作者：
Reich, NO
中科院分区：
文献类型：
--
作者：
Svedruzic, ZM;Reich, NO
为了研究细菌DNA胞嘧啶甲基转移酶M.HhaI的催化机制，我们测定了在有H2Met类似物存在下胞嘧啶C-5上的氚交换反应。聚（dG-dC）和聚（dI-dC）基板被用来研究活性位点环（残基80 - 99）的功能，螺旋外碱基的稳定性，碱基翻转机制，和DNA基板上的持续合成能力。几种实验方法的基础上，我们表明，甲基转移是限速的前稳态步骤。此外，我们表明，活性位点开环有助于在多个循环的催化过程中的限速步骤。靶碱基活化和半胱氨酸81的亲核攻击是快速且容易可逆的。因此，涉及活化的目标碱和螺旋外碱的反应中间体处于平衡状态，并在缓慢的甲基转移步骤之前积累。活化的靶碱基的稳定性取决于活性位点环闭合，其取决于异亮氨酸86和靶胞嘧啶5 '的鸟嘌呤之间的氢键。这些相互作用防止螺旋外碱基的过早释放和不受控制的溶剂进入;后者调节交换反应，并暗示诱变脱氨基反应。M. HhaI的过程催化作用也受异亮氨酸86和DNA底物之间的相互作用调节。当靶碱基在螺旋外位置没有完全稳定时，半胱氨酸81的亲核攻击是部分限速的，如在与Gln（237）Trp突变体反应期间或在不存在辅因子的情况下在胞嘧啶C-5交换反应中所观察到的。
We measured the tritium exchange reaction on cytosine C-5 in the presence of AdoMet analogues to investigate the catalytic mechanism of the bacterial DNA cytosine methyltransferase M.HhaI. Poly(dG-dC) and poly(dI-dC) substrates were used to investigate the function of the active site loop (residues 80-99), stability of the extrahelical base, base flipping mechanism, and processivity on DNA substrates. On the basis of several experimental approaches, we show that methyl transfer is the rate-limiting pre-steady-state step. Further, we show that the active site loop opening contributes to the rate-limiting step during multiple cycles of catalysis. Target base activation and nucleophilic attack by cysteine 81 are fast and readily reversible. Thus, the reaction intermediates involving the activated target base and the extrahelical base are in equilibrium and accumulate prior to the slow methyl transfer step. The stability of the activated target base depends on the active site loop closure, which is dependent on the hydrogen bond between isoleucine 86 and the guanine 5' to the target cytosine. These interactions prevent the premature release of the extrahelical base and uncontrolled solvent access; the latter modulates the exchange reaction and, by implication, the mutagenic deamination reaction. The processive catalysis by M.HhaI is also regulated by the interaction between isoleucine 86 and the DNA substrate. Nucleophilic attack by cysteine 81 is partially rate limiting when the target base is not fully stabilized in the extrahelical position, as observed during the reaction with the Gln(237)Trp mutant or in the cytosine C-5 exchange reaction in the absence of the cofactor.