Mutational and pH studies of the 3′→5′ exonuclease activity of bacteriophage T4 DNA polymerase
Mutational and pH studies of the 3′→5′ exonuclease activity of bacteriophage T4 DNA polymerase
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DOI：
10.1074/jbc.274.35.25151
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发表时间：
1999-08-27
影响因子：
4.8
通讯作者：
Reha-Krantz, LJ
中科院分区：
文献类型：
--
作者：
Elisseeva, E;Mandal, SS;Reha-Krantz, LJ
校对DNA聚合酶的3‘-gt；5’外切酶活性需要两个二价金属离子A和B，对噬菌体T4 DNA聚合酶3‘-gt；5’外切酶活性中心的突变研究表明，与金属离子A结合的残基Asp-324是水解反应最重要的残基。在没有非酶源氢氧离子的情况下，丙氨酸取代残基Asp-324使核酸外切酶活性降低10-100倍，是其他金属结合残基Asp-112和Asp-219的10-100倍，因此，与野生型酶相比，D324A-DNA聚合酶的核酸外切酶活性降低了10(5)倍，而D219A-和D112A/E114A-DNA聚合酶的核酸外切酶活性分别降低了10(3)-10(4)倍。我们的结果与水分子在金属离子A的配位下形成金属氢氧化物离子的观点是一致的，该金属氢氧化物离子的取向是攻击裂解部位的磷酸二酯键。残基Glu-114和Lys-299可能通过降低金属离子-A配位水分子的pk(A)来帮助反应，而残基Tyr-320可能有助于将DNA从结合构象重定向到催化活性构象。
The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B, Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10-100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219, Thus, exonuclease activity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the pK(a) of the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation.