Kinetic properties of Mycobacterium tuberculosis bifunctional GlmU
Kinetic properties of Mycobacterium tuberculosis bifunctional GlmU
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DOI：
10.1007/s00203-011-0715-8
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发表时间：
2011-05
影响因子：
2.8
通讯作者：
Yan Zhou;Y. Xin;Shanshan Sha;Yufang Ma
中科院分区：
文献类型：
--
作者：
Yan Zhou;Y. Xin;Shanshan Sha;Yufang Ma
UDP-N-乙酰葡糖胺（UDP-GlcNAc）作为二糖接头（d-N-GlcNAc-1-鼠李糖）的糖基供体之一和分枝杆菌中肽聚糖的前体存在。双功能酶GlmU参与UDP-GlcNAc合成途径的最后两个连续步骤。葡糖胺-1-磷酸乙酰转移酶催化葡糖胺-1-磷酸（GlcN-1-P）和乙酰辅酶A（乙酰CoA）形成N-乙酰葡糖胺-1-磷酸（GlcNAc-1-P），N-乙酰葡糖胺-1-磷酸尿苷酰转移酶催化GlcNAc-1-P和UTP合成UDP-GlcNAc。先前的研究表明GlmU对分枝杆菌存活的重要性，支持GlmU作为结核病药物的新的和潜在的靶标。本工作建立了两种基于96孔板的比色法，测定了双功能GlmU的动力学性质，包括初速度、最适温度、最适pH、Mg ~（2+）的影响以及动力学参数。双功能GlmU酶活性和动力学性质的比色测定将有助于高通量筛选GlmU抑制剂。
The UDP-N-acetylglucosamine (UDP-GlcNAc) is present as one of the glycosyl donors for disaccharide linker (d-N-GlcNAc-l-rhamnose) and the precursor of peptidoglycan in mycobacteria. The bifunctional enzyme GlmU involves in the last two sequential steps of UDP-GlcNAc synthetic pathway. Glucosamine-1-phosphate acetyltransferase catalyzes the formation of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) from glucosamine-1-phosphate (GlcN-1-P) and acetyl coenzyme A (Acetyl CoA), and N-acetylglucosamine-1-phosphate uridyltransferase catalyzes the synthesis of UDP-GlcNAc from GlcNAc-1-P and UTP. The previous studies demonstrating the essentiality of GlmU to mycobacterial survival supported GlmU as a novel and potential target for TB drugs. In this work, two accurate and simple colorimetric assays based on 96-well microtiter plate were developed to measure the kinetic properties of bifunctional GlmU including initial velocity, optimal temperature, optimal pH, the effect of Mg2+, and the kinetic parameters. Both of the colorimetric assays for bifunctional GlmU enzyme activities and the kinetic properties will facilitate high-throughput screening of GlmU inhibitors.