Residues distal from the active site that alter enzyme function in M.HhaI DNA cytosine methyltransferase.
Residues distal from the active site that alter enzyme function in M.HhaI DNA cytosine methyltransferase.
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远离活性位点的残基会改变 M.HhaI DNA 胞嘧啶甲基转移酶的酶功能。
DOI：
10.1080/07391102.2005.10507023
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发表时间：
2005
影响因子：
4.4
通讯作者：
Reich,Norbert
中科院分区：
文献类型：
--
作者：
Sharma,Vyas;Youngblood,Ben;Reich,Norbert
用丙氨酸替换10个M.HhaI残基，以探测远端蛋白元件对底物/辅因子结合21、甲基转移和产物释放的重要性。通过热力学分析、预稳态和稳态动力学评估从活性位点6-20 μ m范围内的取代，以获得KdMet、KdDNA、kcat/KmDNA、kcat和kmethyltransfervalues。对于野生型M.HhaI，产物释放步骤主导催化营业额，而4倍更快的内部微观常数kmethyltransferpresents一个上限。甲基转移反应的δH ε和δS ε值分别为10.3 kcal/mol和-29.4 cal/（mol K），与气相中观察到的压缩过渡态相似。虽然10个突变体在甲基转移中基本上保持不变，但观察到影响底物/辅因子结合和产物释放的长期效应。在Asp 73 Ala和Val 282 Ala中观察到阳性增强，Asp 73 Ala显示出对Met的亲和力提高了25倍，Val 282 Ala显示出催化转化率提高了4倍。基于C5-胞嘧啶DNA甲基转移酶家族内的位置概率的分析，我们提出，某些保守的远端残基可能是重要的，在介导的长期影响。
Ten M.HhaI residues were replaced with alanine to probe the importance of distal protein elements to substrate/cofactor binding21,methyl transfer, and product release. The substitutions, ranging from 6–20 Å from the active site were evaluated by thermodynamic analysis, pre-steady and steady-state kinetics, to obtain KdAdoMet, KdDNA, kcat/KmDNA, kcat, and kmethyltransfervalues. For the wild-type M.HhaI, product release steps dominate catalytic turnover while the 4-fold faster internal microscopic constant kmethyltransferpresents an upper limit. The methyl transfer reaction has δH‡ and δS‡ values of 10.3 kcal/mol and—29.4 cal/(mol K), respectively, consistent with a compressed transition state similar to that observed in the gas phase. Although the ten mutants remained largely unperturbed in methyl transfer, long-range effects influencing substrate/cofactor binding and product release were observed. Positive enhancements were seen in Asp73Ala, which showed a 25fold improvement in AdoMet affinity and in Val282Ala, which showed a 4-fold improvement in catalytic turnover. Based on an analysis of the positional probability within the C5- cytosine DNA methyltransferase family we propose that certain conserved distal residues may be important in mediating long-range effects.