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首页> 外文期刊>Biochemistry >Bronsted Analysis and Rate-Limiting Steps for the T5 Flap Endonuclease Catalyzed Hydrolysis of Exonucleolytic Substratest
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Bronsted Analysis and Rate-Limiting Steps for the T5 Flap Endonuclease Catalyzed Hydrolysis of Exonucleolytic Substratest

机译：用于T5瓣内切核酸酶催化水解的抗正囊分析及速率限制步骤

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摘要

During replication and repair flap endonucleases (FENs) catalyze endonucleolytic and exonucleolytic (EXO) DNA hydrolyses. Altering the leaving group pK_a, by replacing the departing nucleoside with analogues, had minimal effect on k_(cat)/K_M in a T5FEN-catalyzed EXO reaction, producing a very low Bronsted coefficient,β_(lg). Investigation of the viscosity dependence of k_(cat)/K_M revealed that reactions of EXO substrates are rate limited by diffusional encounter of enzyme and substrate, explaining the small β_(lg).However, the maximal single turnover rate of the FEN EXO reaction also yields a near zero β_(lg). A low β_(lg), was also observed when evaluating k_(cat_/K_M for D2011/D204S FEN-catalyzed reactions, even though these reactions were not affected by added viscogen. But an active site K83A mutant produced a β_(lg)= -1.2 + 0.10, closer to the value observed for solution hydrolysis of phosphate diesters. The pH—maximal rate profiles of the WT and K83A FEN reactions both reach a maximum at high pH and do not support an explanation of the data that involves catalysis of leaving group departure by Lys 83 functioning as a general acid. Instead, a ratelimiting physical step, such as substrate unpairing or helical arch ordering, that occurs after substrate association must kinetically hide an inherent large β_(lg). It is suggested that K83 acts as an electrostatic catalyst that stabilizes the transition state for phosphate diester hydrolysis. When K83 is removed from the active site,chemistry becomes rate limiting and the leaving group sensitivity of the FEN-catalyzed reaction is revealed.
机译：在复制和修复皮瓣内切核酸酶（FICS）期间催化内切核酸酐和外核酸溶解（EXO）DNA水解。通过用类似物替换离去的核苷来改变离去组PK_A，对T5FEN催化的外部反应中的K_（猫）/ Km具有最小的影响，产生非常低的布朗斯系数β_（LG）。 k_（猫）/ k_m的粘度依赖性的研究表明，EXO底物的反应是通过酶和底物扩散腹泻的速率限制，解释了小β_（lg）。然而，芬汁反应的最大单个周转率也是如此产生接近零β_（LG）。当评估K_（对于D2011 / D204S Fen催化反应的CAT_ / K_M时，也观察到低β-（LG），即使这些反应不受添加的粘胰剂的影响。但是活性位点K83A突变体产生β_（LG）= -1.2 + 0.10，接近磷酸盐二酯溶液水解的值。WT和K83A FEN反应的pH最大速率曲线均在高pH下达到最大值，并且不支持涉及催化的数据的解释通过Lys 83离开群体偏离作为一般性酸。相反，基板关联之后发生的标准物理步骤，例如基板未配位或螺旋拱命令，必须动态隐藏固有的大β_（LG）。建议K83作用作为静电催化剂，其稳定磷酸二酯水解的过渡状态。当从活性位点移除K83时，化学变为速率限制，并揭示了芬催化反应的离开组敏感性。

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  • 来源
    《Biochemistry》 |2010年第37期|共9页
  • 作者

    Blanka Sengerova; Christopher Tomlinson; John M. Atack; Ryan Williams; Jon R. Sayers; Nicholas H. Williams; Jane A. Grasby;

  • 作者单位

    Centre fir Chemical Biology Department of Chemistry University of Sheffield Sheffield S3 7HF U.K.;

    Centre fir Chemical Biology Department of Chemistry University of Sheffield Sheffield S3 7HF U.K.;

    Centre fir Chemical Biology Department of Chemistry University of Sheffield Sheffield S3 7HF U.K.;

    Centre fir Chemical Biology Department of Chemistry University of Sheffield Sheffield S3 7HF U.K.;

    Henry Wellcome Lahoratoriesfor Medical Research. University of Sheffield School of Medicine and Biomedical Science Beech Hill Road. Sheffield S10 2RX U.K.;

    Centre fir Chemical Biology Department of Chemistry University of Sheffield Sheffield S3 7HF U.K.;

    Centre fir Chemical Biology Department of Chemistry University of Sheffield Sheffield S3 7HF U.K.;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

    replication; diffusional; maximum;

    机译：复制;扩散;最大;

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