Mapping enzyme active sites in complex proteomes
Mapping enzyme active sites in complex proteomes
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DOI：
10.1021/ja038441g
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发表时间：
2004-02-11
影响因子：
15
通讯作者：
Cravatt, BF
中科院分区：
文献类型：
--
作者：
Adam, GC;Burbaum, J;Cravatt, BF
基因组测序项目已经发现了许多新的酶和酶类，其活性位点结构和机制的知识是有限的。为了促进原核和真核基因组编码的众多酶的机制研究，需要新的方法来分析高生物复杂性样品中的酶功能。在这里，我们描述了一个通用的策略，利用活性为基础的化学探针加上一个无凝胶的分析平台，在整个蛋白质组中的酶活性位点的分析。我们应用这种无凝胶的策略来确定磺酸酯探针靶向酶的标记位点。对于所检查的每种酶，发现探针标记发生在保守的活性位点残基上，包括催化亲核试剂（例如，谷胱甘肽S-转移酶ω中的C32）和碱/酸（例如，醛脱氢酶-1中的E269;烯酰CoA水合酶-1中的D204），以及功能未知的残基（例如，3 β-羟基类固醇脱氢酶/异构酶-1中的D127）。这些结果表明，磺酸酯探针是非常通用的基于活性的分析试剂，能够标记多种催化残基的范围内的机械不同的酶。更一般地，本文所述的无凝胶策略通过将基于活性的探针的蛋白质靶标和由这些试剂标记的特异性残基的鉴定合并成单个步骤，提供了一种新的平台，其中酶的蛋白质组学比较可以与其活性位点的机制分析一致地完成。
Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel-free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3beta-hydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites.