Mechanistic insight into 3-deoxy-D-manno-octulosonate-8-phosphate synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase utilizing phosphorylated monosaccharide analogues.
Mechanistic insight into 3-deoxy-D-manno-octulosonate-8-phosphate synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase utilizing phosphorylated monosaccharide analogues.
复制标题
利用磷酸化单糖类似物对 3-脱氧-D-甘露-辛糖酸-8-磷酸合酶和 3-脱氧-D-阿拉伯-庚糖酸-7-磷酸合酶的机制进行深入了解。
DOI：
10.1021/bi026553z
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发表时间：
2003
期刊：
影响因子：
2.9
通讯作者：
Woodard,RonaldW
中科院分区：
文献类型：
--
作者：
Howe,DavidL;Sundaram,AppavuK;Wu,Jing;Gatti,DomenicoL;Woodard,RonaldW
大肠埃希氏菌3-脱氧-d-甘露糖8-磷酸(KDO8-P)合成酶能利用5碳的磷酸化单糖2-脱氧核糖-5-磷酸(2dR5P)作为替代底物，而不能利用5-磷酸核糖(R5P)或4碳类似物-4-磷酸(E4P)。然而，E.coliKDO8-P合成酶在R5P或E4P存在的情况下，催化每个活性部位快速消耗约1摩尔的PEP，之后PEP的消耗放缓至可忽略但可测量的速度。用[2，3-13C2]-PEP和[3-F]-PEP研究了PEP流产的机理，并用~(13)C、~(31)P和~(19)F核磁共振确定反应产物为丙酮酸、磷酸和2-磷酸甘油酸(2-PGA)。丙酮酸和2-PGA的形成表明，KDO8-P合成酶催化的反应可能是通过水分子对PEP的亲核攻击而启动的。在同源酶3-脱氧-d-阿拉伯-庚磺酸盐7-磷酸(DAH7-P)与D孵育的实验中，L-甘油醛3-磷酸(G3P)和[2，3-13C2]-PEP形成的主要物种是丙酮酸和磷酸，这表明DAH7-P合成酶催化的反应是从水对PEP的亲核攻击开始的。ColiKDO8-P合成酶。
Escherichia coli3-deoxy-d-manno-octulosonate 8-phosphate (KDO8-P) synthase is able to utilize the five-carbon phosphorylated monosaccharide, 2-deoxyribose 5-phosphate (2dR5P), as an alternate substrate, but notd-ribose 5-phosphate (R5P) nor the four carbon analogued-erythrose 4-phosphate (E4P). However,E. coliKDO8-P synthase in the presence of either R5P or E4P catalyzes the rapid consumption of approximately 1 mol of PEP per active site, after which consumption of PEP slows to a negligible but measurable rate. The mechanism of this abortive utilization of PEP was investigated using [2,3-13C2]-PEP and [3-F]-PEP, and the reaction products were determined by13C,31P, and19F NMR to be pyruvate, phosphate, and 2-phosphoglyceric acid (2-PGA). The formation of pyruvate and 2-PGA suggests that the reaction catalyzed by KDO8-P synthase may be initiated via a nucleophilic attack to PEP by a water molecule. In experiments in which the homologous enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH7-P) synthase was incubated withd,l-glyceraldehyde 3-phosphate (G3P) and [2,3-13C2]-PEP, pyruvate and phosphate were the predominant species formed, suggesting that the reaction catalyzed by DAH7-P synthase starts with a nucleophilic attack by water onto PEP as observed inE. coliKDO8-P synthase.