Syson Karl, Tomlinson Christopher, Chapados Brian R, Sayers Jon R, Tainer John A, Williams Nicholas H, Grasby Jane A
Department of Chemistry, Centre for Chemical Biology, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom.
J Biol Chem. 2008 Oct 17;283(42):28741-6. doi: 10.1074/jbc.M801264200. Epub 2008 Aug 11.
Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a "two-metal-ion mechanism." Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of > 4 A appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2+ or Mn2+). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal-dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions.
蛋白质核酸酶和RNA酶在DNA复制、DNA修复、RNA加工和RNA降解过程中依赖二价金属离子来催化核酸磷酸二酯键的快速水解。人们普遍认为这些酶通过“双金属离子机制”催化磷酸二酯水解。然而，对翼状内切核酸酶（FEN）家族成员的分析表明，对于磷酸二酯水解的经典双金属离子机制存在争议。FEN家族成员存在于生命的所有领域，在DNA复制和修复中发挥作用。虽然FEN的无底物结构确定了两个活性位点金属离子，但它们典型的大于4 Å的间距似乎与该机制不兼容。为了阐明FEN金属离子所起的作用，我们在此报告了对T5FEN镁离子反应的详细评估。动力学研究表明，总体而言，T5FEN催化的反应至少需要三个镁离子，这意味着还结合了一个额外的金属离子。催化磷酸二酯水解需要存在至少两个具有不同亲和力结合的离子。对钙离子对反应抑制作用的分析与需要两种可行的辅因子（Mg2+或Mn2+）一致。通过结合两个镁离子，表观底物缔合常数最大化。这可能反映了双链底物的金属依赖性解链，以使可切割的磷酸与金属离子接触。综合结果表明，T5FEN主要使用双金属离子机制进行化学催化，但其整体金属生物化学更为复杂，需要三个离子。
