Structural and functional comparisons of nucleotide pyrophosphatase/phosphodiesterase and alkaline phosphatase: Implications for mechanism and evolution
Structural and functional comparisons of nucleotide pyrophosphatase/phosphodiesterase and alkaline phosphatase: Implications for mechanism and evolution
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DOI：
10.1021/bi060847t
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发表时间：
2006-08-15
期刊：
影响因子：
2.9
通讯作者：
Herschlag, Daniel
中科院分区：
文献类型：
--
作者：
Zalatan, Jesse G.;Fenn, Timothy D.;Herschlag, Daniel
基因组和结构数据量的快速扩展提供了许多具有催化不同反应的进化相关活性位点的酶的例子。这些活性位点的功能比较可以提供洞察酶的巨大催化能力的起源和可能导致不同酶活性的进化变化。碱性磷酸酶（AP）超家族是一个理想的系统，用于进行这样的比较，给出了广泛的数据上的非酶和酶的磷酰基转移反应。一些超家族成员，如AP本身，优先水解磷酸单酯，而其他成员，如核苷酸焦磷酸酶/磷酸二酯酶（NPP），优先水解磷酸二酯。我们已经测量了NPP催化的磷酸二酯和单酯水解的速率常数。根据二酯底物的特性，NPP以10（2）-10（6）的因子优先催化二酯水解。为了确定可能导致优先磷酸二酯水解的NPP活性位点的特征，我们已经确定了在没有配体的情况下以及在与钒酸盐和AMP的复合物中NPP的结构。与AP现有结构的比较揭示了结构上无法区分的双金属核心，但在保守的双金属位点之外有几个不同的结构特征。结构和功能数据一起表明，这些不同的官能团中的一些提供特定的底物结合相互作用，而另一些则调整双金属活性位点本身的性质，以区分磷酸二酯和单酯底物。
The rapid expansion of the amount of genomic and structural data has provided many examples of enzymes with evolutionarily related active sites that catalyze different reactions. Functional comparisons of these active sites can provide insight into the origins of the enormous catalytic proficiency of enzymes and the evolutionary changes that can lead to different enzyme activities. The alkaline phosphatase (AP) superfamily is an ideal system to use in making such comparisons given the extensive data available on both nonenzymatic and enzymatic phosphoryl transfer reactions. Some superfamily members, such as AP itself, preferentially hydrolyze phosphate monoesters, whereas others, such as nucleotide pyrophosphatase/phosphodiesterase (NPP), preferentially hydrolyze phosphate diesters. We have measured rate constants for NPP-catalyzed hydrolysis of phosphate diesters and monoesters. NPP preferentially catalyzes diester hydrolysis by factors of 10(2)-10(6), depending on the identity of the diester substrate. To identify features of the NPP active site that could lead to preferential phosphate diester hydrolysis, we have determined the structure of NPP in the absence of ligands and in complexes with vanadate and AMP. Comparisons to existing structures of AP reveal bimetallo cores that are structurally indistinguishable, but there are several distinct structural features outside of the conserved bimetallo site. The structural and functional data together suggest that some of these distinct functional groups provide specific substrate binding interactions, whereas others tune the properties of the bimetallo active site itself to discriminate between phosphate diester and monoester substrates.