The kinetic characterization of Escherichia coli MurG using synthetic substrate analogues
The kinetic characterization of Escherichia coli MurG using synthetic substrate analogues
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DOI：
10.1021/ja991556t
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发表时间：
1999-09-22
影响因子：
15
通讯作者：
Walker, S
中科院分区：
文献类型：
--
作者：
Ha, S;Chang, E;Walker, S
细菌对现有抗生素的耐药性对人类健康构成严重威胁。由于细菌细胞周围的肽聚糖层是生存所必需的，因此参与肽聚糖生物合成的酶是设计新抗生素的诱人靶标。不幸的是，这些酶中的许多很难研究，因为在合适的检测条件下，无法获得或不能溶解监测酶活性的底物。这些问题可以通过使用合成的替代底物来解决。我们最近报道了MURG的可溶性底物类似物的合成，MURG是形成肽多聚糖的beta-(1，4)-N-acetylglucosaminyl-N-acetylmuramyl五肽亚单位的酶。使用这种底物类似物，我们已经能够开发一种直接的方法来监测酶的活性。我们现在报道了大肠杆菌Murg的纯化，以及在无膜条件下其动力学性质和底物需求的信息。这项工作为这种重要的细菌酶的详细机制和结构研究奠定了基础。
Bacterial resistance to existing antibiotics poses a serious threat to human health. Because the peptidoglycan layer surrounding bacterial cells is essential for survival, the enzymes involved in peptidoglycan biosynthesis are attractive targets for the design of new antibiotics. Unfortunately, many of these enzymes are difficult to study because substrates to monitor enzymatic activity are either not available or not soluble under suitable assay conditions. These problems can be solved by utilizing synthetic alternative substrates. We recently reported the synthesis of a soluble substrate analogue for MurG, the enzyme that forms the beta-(1,4)-N-acetylglucosaminyl-N-acetylmuramyl pentapeptide subunit of peptidoglycan. Using this substrate analogue, we have been able to develop a direct assay to monitor the activity of the enzyme. We-now report the purification of Escherichia coli MurG and information on its kinetic properties and substrate requirements in the absence of membranes. This work lays the foundation for detailed mechanistic and structural investigations of this essential bacterial enzyme.