Nucleotide pyrophosphatase employs a P-loop-like motif to enhance catalytic power and NDP/NTP discrimination
Nucleotide pyrophosphatase employs a P-loop-like motif to enhance catalytic power and NDP/NTP discrimination
复制标题
DOI：
10.1073/pnas.1013872108
复制
发表时间：
2011-08-30
影响因子：
11.1
通讯作者：
Toth, Judit
中科院分区：
文献类型：
--
作者：
Pecsi, Ildiko;Szabo, Judit E.;Toth, Judit
我们研究了核苷酸焦磷酸酶中潜在的（d）NDP/（d）NTP识别机制。在这里，我们报告说，dUTPase是一种必需的核苷酸焦磷酸酶，它以独特的机制使用C末端P环样序列来区分底物和有效水解。我们对人dUTR突变体的光谱和瞬态动力学结果结合以前的结构研究表明：（i）γ-磷酸和P-环样基序V之间的氢键相互作用促进了α-磷酸基团处反应中心的催化活性构象;（ii）这些相互作用加速了动力学循环的化学步骤，并且（iii）在不存在γ-磷酸盐的情况下，水解非常缓慢地发生或根本不发生。基序V相互作用，即，在dUDP、dUDP.BeFx中，或在基序V缺失突变体中。dUTP的生理作用是设定细胞dUTP：dTTP比率并防止有害的尿嘧啶掺入DNA。基于与相关的焦磷酸生成（d）NTPases的比较，我们提出，不寻常的使用的P-环样基序使dUTPases能够实现有效的催化dUTP水解和有效的歧视dUDP在同一时间。这些特性可能对尿嘧啶-DNA修复的出现有利。dUTR基序V和P-环（或步行者A序列）之间的相似性和差异通常由ATP酶和GTP酶表征，提供了对各种核苷酸水解任务的功能适应的深入了解。
We investigated the potential (d)NDP/(d)NTP discrimination mechanisms in nucleotide pyrophosphatases. Here, we report that dUTPase, an essential nucleotide pyrophosphatase, uses a C-terminal P-loop-like sequence in a unique mechanism for substrate discrimination and efficient hydrolysis. Our spectroscopy and transient kinetics results on human dUTPase mutants combined with previous structural studies indicate that (i) H-bond interactions between the gamma-phosphate and the P-loop-like motif V promote the catalytically competent conformation of the reaction center at the alpha-phosphate group; (ii) these interactions accelerate the chemical step of the kinetic cycle and that (iii) hydrolysis occurs very slowly or not at all in the absence of the gamma-phosphate-motif V interactions, i.e., in dUDP, dUDP.BeFx, or in the motif V-deleted mutant. The physiological role of dUTPase is to set cellular dUTP: dTTP ratios and prevent injurious uracil incorporation into DNA. Based upon comparison with related pyrophosphate generating (d)NTPases, we propose that the unusual use of a P-loop-like motif enables dUTPases to achieve efficient catalysis of dUTP hydrolysis and efficient discrimination against dUDP at the same time. These specifics might have been advantageous on the appearance of uracil-DNA repair. The similarities and differences between dUTPase motif V and the P-loop (or Walker A sequence) commonly featured by ATP- and GTPases offer insight into functional adaptation to various nucleotide hydrolysis tasks.