Kinetic characterization of wild-type and S229A mutant MurB: Evidence for the role of ser 229 as a general acid
Kinetic characterization of wild-type and S229A mutant MurB: Evidence for the role of ser 229 as a general acid
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DOI：
10.1021/bi962220o
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发表时间：
1997-01-28
期刊：
影响因子：
2.9
通讯作者：
Massey, V
中科院分区：
文献类型：
--
作者：
Benson, TE;Walsh, CT;Massey, V
X射线结构数据预测，丝氨酸229在细菌肽聚糖生物合成黄素酶MurB催化下还原烯醇式丙酮-UDP-N-乙酰氨基葡萄糖的过程中，被定位为正在发育的C2碳负离子的质子供体。为了研究这种影响，构建并纯化了一个用丙氨酸取代丝氨酸229的突变体，对突变酶的两个半反应的动力学分析表明，NADPH对EFL(Ox)的还原减少了9倍，EF(RED)与烯醇式丙酮酸底物的再氧化作用显著减少了10(7)倍。此外，对S229A与底物类似物(E)-烯醇丁酰基-UDP-N-乙酰氨基葡萄糖的研究表明，分配倾向于形成(Z)几何异构体，而不是形成还原产物UDP-甲基咪唑酸，这是野生型MurB的主要产物。这些研究为该活性中心丝氨酸作为普通酸催化剂的作用提供了证据。
X-ray derived structural data predicted that serine 229 was positioned to act as a proton donor to the developing C2 carbanion during the reduction of enolpyruvyl-UDP-N-acetylglucosamine catalyzed by the bacterial peptidoglycan biosynthetic flavoenzyme MurB. To investigate this effect, a mutant where serine 229 was replaced by alanine was constructed and purified, Kinetic analysis of the two half-reactions for the mutant enzyme revealed a 9-fold decrease in the reduction of EFl(ox) by NADPH and a dramatic 10(7)-fold decrease in the reoxidation of EF(red) with the enolpyruvyl substrate, In addition, studies of S229A with the substrate analog, (E)-enolbutyryl-UDP-N-acetylglucosamine, showed a striking bias of the partitioning toward formation of the (Z) geometric isomer as opposed to formation of the reduced product UDP-methylmuramic acid, which was the predominant product in wild-type MurB. These studies provide evidence for the proposed role of this active-site serine as a general acid catalyst.