EVIDENCE THAT THE REACTION OF THE UDP-N-ACETYLGLUCOSAMINE 1-CARBOXYVINYLTRANSFERASE PROCEEDS THROUGH THE O-PHOSPHOTHIOKETAL OF PYRUVIC-ACID BOUND TO CYS115 OF THE ENZYME
EVIDENCE THAT THE REACTION OF THE UDP-N-ACETYLGLUCOSAMINE 1-CARBOXYVINYLTRANSFERASE PROCEEDS THROUGH THE O-PHOSPHOTHIOKETAL OF PYRUVIC-ACID BOUND TO CYS115 OF THE ENZYME
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DOI：
10.1111/j.1432-1033.1993.tb18442.x
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发表时间：
1993-12-15
期刊：
影响因子：
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通讯作者：
AMRHEIN, N
中科院分区：
文献类型：
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作者：
WANKE, C;AMRHEIN, N
酶UDP- n -乙酰氨基葡萄糖1-羧乙烯基转移酶（enolpyruvyltransferase, EC 2.5.1.7）催化磷酸烯醇丙酮酸的完整的1-羧乙烯基片段转移到UDP-(2')- n -乙酰氨基葡萄糖胺的3'-羟基上，同时释放无机磷酸盐，这是细菌细胞壁肽聚糖生物合成的第一步。从阴沟肠杆菌中提取的酶在大肠杆菌中过表达，可以分离出大量的纯化酶(约为1。900毫克/20克新鲜菌群)。通过将酶与c -14标记的磷酸烯醇丙酮酸、p -32标记的正磷酸盐和未标记的UDP-（2’）- n -乙酰-（3’）-1-羧乙烯基葡萄糖胺孵育，我们能够分离并表征一种与蛋白质共价结合的反应中间体。它含有相对于蛋白质的磷酸烯醇丙酮酸的C3部分（0.98 mol/mol）和磷酸部分（0.95 mol/mol）的化学计量量。在UDP-(2')- n-乙酰氨基葡萄糖存在的情况下，这种蛋白质结合的中间体向产物UDP-(2')- n-乙酰-(3')-1-羧基乙烯基氨基葡萄糖的快速周转表明该中间体具有动力学能力。我们也有证据表明中间体作为丙酮酸的0-磷酸硫酮结合到酶的Cys115上。磷霉素是一种不可逆的UDP-GlcNAc羧乙烯基转移酶抑制剂，它与同样的胱氨酸残基共价结合。Cys115与丝氨酸残基交换导致酶失活，证明了Cys115在反应中的重要作用。已知唯一催化磷酸烯醇丙酮酸完整的1-羧乙烯基部分转移到底物的酶是3-磷酸莽草酸1-羧乙烯基转移酶（EC 2.5.1.19），它是莽草酸途径的第6种酶。已知这种合酶的反应是通过一个单一的，紧密的但不是共价结合的四面体中间体进行的。尽管这两种酶在初级氨基酸序列上有相似之处，但它们的反应机制似乎有本质上的不同。
The enzyme UDP-N-acetylglucosamine 1-carboxyvinyltransferase (enolpyruvyltransferase, EC 2.5.1.7) catalyses the transfer of the intact 1-carboxyvinyl moiety of phosphoenolpyruvate to the 3'-hydroxyl group of the glucosamine moiety of UDP-(2')-N-acetylglucosamine with the concomitant release of inorganic phosphate, the first committed step in the biosynthesis of the bacterial cell wall peptidoglycan. Overexpression of the enzyme from Enterobacter cloacae in Escherichia coli allowed the isolation of large amounts of purified enzyme (approx. 900 mg/20 g fresh mass bacteria). By incubating the enzyme with C-14-labelled phosphoenolpyruvate, P-32-labelled orthophosphate and unlabelled UDP-(2')-N-acetyl-(3')-1-carboxyvinylglucosamine, we were able to isolate and characterise a reaction intermediate, covalently bound to the protein. It contains stoichiometric quantities of the C3 moiety (0.98 mol/mol) as well as of the phosphate moiety (0.95 mol/mol) of phosphoenolpyruvate relative to protein. The rapid turnover of this protein-bound intermediate in the presence of UDP-(2')-N-acetylglucosamine towards the product UDP-(2')-N-acetyl-(3')-1-carboxyvinylglucosamine suggests that the intermediate is kinetically competent. We also present evidence that the intermediate is bound as the 0-phosphothioketal of pyruvic acid to Cys115 of the enzyme. This is the same Cys residue to which phosphomycin, an irreversible inhibitor of the UDP-GlcNAc carboxyvinyltransferase, binds covalently. Exchange of Cys115 for a Ser residue resulted in an inactive enzyme, demonstrating the essential role of Cys115 for the reaction.The only other enzyme known to catalyse the transfer of the intact 1-carboxyvinyl moiety of phosphoenolpyruvate to a substrate is the 3-phosphoshikimate 1-carboxyvinyltransferase (EC 2.5.1.19), die sixth enzyme of the shikimate pathway. The reaction of this synthase is known to proceed through a single, tightly but not covalently bound, tetrahedral intermediate. Even though the two enzymes share-similarities in their primary amino acid sequences, their reaction mechanisms appear to be substantially different.