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首页> 外文期刊>Biochemistry >Effect of pH on the Active Site of an Argl21Cys Mutant of the Metallo-beta-lactamase from Bacillus cereus:Implications for the Enzyme Mechanism
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Effect of pH on the Active Site of an Argl21Cys Mutant of the Metallo-beta-lactamase from Bacillus cereus:Implications for the Enzyme Mechanism

机译：pH对芽孢杆菌腺嘌呤β-内酰胺酶的arg121cys突变体活性位点的影响：对酶机制的影响

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摘要

The zinc-dependent metallo-beta-lactamases are a group of bacterial enzymes that pose a threat to the future efficacy of present-day antibiotics.Their mechanism is poorly understood,and there are no clinically useful inhibitors.While most members of the group contain two tightly bound zinc ions in their active sites,the Bacillus cereus enzyme has a much lower affinity for its second zinc(Zn2),thought to be due to the presence of Argl21 immediately beneath the floor of the active site(cf.Cys/Ser/Hisl21 in the bizinc enzymes).Crystal structures of the Argl21Cys mutant of the B.cereus 569/H/9 enzyme were solved at pH 7.0,5.0,and 4.5,each in the presence of either 20 muM or 20 mM Zn~(2+) to generate the mono- and bizinc forms,respectively.Surprisingly,the structure of the active site was unaffected by the mutation;a network of ordered water molecules replaced the interactions made by the arginine side chain,and the occupancy of Zn2 appeared minimally changed.As the pH was lowered,Zn2 moved away from one of its ligands,Asp 120,but was "tracked" by "two others,Cys221 and His263.Furthermore,the hydroxide ion(and proposed nucleophile for beta-lactam hydrolysis) was bound to Znl at pH 5 and above but absent at pH 4.5.This provides experimental evidence for an earlier proposed mechanism in which protonation of Asp 120 and the Znl-bound hydroxide are the two events that lead to the loss of activity at low pH.
机译：依赖于锌的金属β-内酰胺酶是一组细菌酶，对本日抗生素的未来疗效构成威胁。该机制较差，并且没有临床有用的抑制剂。大多数本集团成员含有在其活性位点中的两个紧密结合的锌离子，芽孢杆菌脑酶对其第二锌（Zn2）具有更低的亲和力，认为是由于活性位点的地板下方的argl21存在（cf.cys / ser / his121在bizinc酶中）。B.Cereus 569 / h / 9酶的arg121cys突变体的晶体结构在pH 7.0,5.0和4.5时溶解在20毫米或20mm Zn的存在下〜（ 2+）分别产生单声道和bizinc的形式。静音，活性部位的结构不受突变的影响;有序水分分子网络取代了精氨酸侧链的相互作用，Zn2的占用率呈现出来最小变化。Zn2移动了pH值远离其一个配体，ASP 120，但是“另外两种，Cys221和His263”被“跟踪”。次摩托，氢氧化物离子（并提出的β-内酰胺水解的亲核试核官）在pH5及以上的Zn1中结合，但不存在在pH 4.5.本质提供了先前提出的机制的实验证据，其中ASP 120和ZnL结合的氢氧化物的质子化是导致低pH下活性损失的两种事件。

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  • 来源
    《Biochemistry》 |2005年第12期|共9页
  • 作者

    Anna M.Davies; Rodolfo M.Rasia; Alejandro J.Vila; Brian J.Sutton; Stella M.Fabiane;

  • 作者单位

    Randall Division of Cell and Molecular Biophysics King's College London New Hunt's House Guy's Campus London Bridge SE1 1UL London United Kingdom and Molecular Biology Division IBR(Instituto de Biologia Molecular y Celular de Rosario) CONICET and Bio;

    Randall Division of Cell and Molecular Biophysics King's College London New Hunt's House Guy's Campus London Bridge SE1 1UL London United Kingdom and Molecular Biology Division IBR(Instituto de Biologia Molecular y Celular de Rosario) CONICET and Bio;

    Randall Division of Cell and Molecular Biophysics King's College London New Hunt's House Guy's Campus London Bridge SE1 1UL London United Kingdom and Molecular Biology Division IBR(Instituto de Biologia Molecular y Celular de Rosario) CONICET and Bio;

    Randall Division of Cell and Molecular Biophysics King's College London New Hunt's House Guy's Campus London Bridge SE1 1UL London United Kingdom and Molecular Biology Division IBR(Instituto de Biologia Molecular y Celular de Rosario) CONICET and Bio;

    Randall Division of Cell and Molecular Biophysics King's College London New Hunt's House Guy's Campus London Bridge SE1 1UL London United Kingdom and Molecular Biology Division IBR(Instituto de Biologia Molecular y Celular de Rosario) CONICET and Bio;

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  • 正文语种 eng
  • 中图分类 生物化学;
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