Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase
Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase
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DOI：
10.1074/jbc.m406135200
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发表时间：
2004-10-08
影响因子：
4.8
通讯作者：
Vertessy, BG
中科院分区：
文献类型：
--
作者：
Barabás, O;Pongrácz, V;Vertessy, BG
脱氧尿苷酶对于将尿嘧啶排除在DNA之外至关重要。测定了野生型和突变型dUTP酶的底物（dUTP和α，β-亚氨基-dUTP）和产物复合物的晶体结构，以揭示负责DNA完整性的酶如何发挥作用。对野生型和突变型dUTPases进行动力学分析以获得溶液中的相关机制信息。底物水解被证明是启动通过在线亲核攻击的水分子由一个激活保守的天冬氨酸残基。底物在催化活性构象中的结合是通过以下方式实现的：（i）三磷酸部分与催化辅助Mg 2+的多重相互作用，（ii）与脱辅基酶相比，来自三个保守酶基序的残基的协同运动，以及（iii）在活性位点中包括几个水分子的复杂氢键网络。结果提供了一个理解的dUTPases中的保守残基的催化作用。
dUTPase is essential to keep uracil out of DNA. Crystal structures of substrate (dUTP and alpha,beta-imino-dUTP) and product complexes of wild type and mutant dUTPases were determined to reveal how an enzyme responsible for DNA integrity functions. A kinetic analysis of wild type and mutant dUTPases was performed to obtain relevant mechanistic information in solution. Substrate hydrolysis is shown to be initiated via in-line nucleophile attack of a water molecule oriented by an activating conserved aspartate residue. Substrate binding in a catalytically competent conformation is achieved by (i) multiple interactions of the triphosphate moiety with catalysis-assisting Mg2+, (ii) a concerted motion of residues from three conserved enzyme motifs as compared with the apoenzyme, and (iii) an intricate hydrogen-bonding network that includes several water molecules in the active site. Results provide an understanding for the catalytic role of conserved residues in dUTPases.