Insights into the mechanism of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (Phe) from Escherichia coli using a transient kinetic analysis
Insights into the mechanism of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (Phe) from Escherichia coli using a transient kinetic analysis
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DOI：
10.1074/jbc.m404753200
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发表时间：
2004-10-29
影响因子：
4.8
通讯作者：
Anderson, KS
中科院分区：
文献类型：
--
作者：
Furdui, C;Zhou, L;Anderson, KS
大肠杆菌苯丙氨酸敏感的3-脱氧-阿拉伯七磺酸7-磷酸合成酶(DAHP合成酶)催化磷酸烯醇式丙酮酸和4-磷酸的羟醛缩合生成3-脱氧-D-阿拉伯-7-磷酸和无机磷。首次报道了大肠杆菌苯丙氨酸敏感的DAHP合成酶的稳态前动力学分析。比较了锰(II)、铜(II)、锌(II)重组DAHP合成酶的稳态和稳态前动力学参数。这些研究表明：1)在生理条件下，三种金属离子的产物释放都是限速的，2)含金属的DAHP合酶活性中心浓度从30%增加到52%，再增加到铜-DAHP合酶的88%，3)在Mn-(130-200 S(-1))中产物形成的速率常数高于铜-(55 S(-1))和锌-DAHP合酶(6.8 S(-1))；(4)锰的稳态速率(产物释放速率常数)(70 S(-1))高于铜(5.6S(-1))和锌-DAHP合酶(1.8S(-1))。此外，在较低pH下的反应动力学研究表明，对于铜-DAHP合成酶，产物的释放不再是限速的，而对于锰和锌-DAHP合成酶，产物的形成速度变慢，这表明中间产物的形成在产物的形成中变得限速。此外，在pH为6.0时，对Mn-DAHP合成酶产物形成的爆裂速率常数也观察到了氚同位素效应。这支持了这样一种假设，即金属离子在大肠杆菌DAHP合成酶中的作用是将氨基酸定位在与水分子配位和激活所需的适当几何形状上。
Escherichia coli phenylalanine-sensitive 3-deoxy-arabinoheptulosonate 7-phosphate synthase (DAHP synthase) catalyzes the net aldol condensation of phosphoenolpyruvate and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate and inorganic phosphate. For the first time, the presteady-state kinetic analysis of the Phe-sensitive DAHP synthase from E. coli is reported. The steady-state and presteady-state kinetic parameters of the DAHP synthase reconstituted with Mn(II), Cu(II), and Zn(II) were compared. These studies showed the following: 1) product release is rate-limiting for all of the three metal ions studied under physiologically relevant conditions; 2) concentration of the active sites of the metal-containing DAHP synthase is increasing from Mn-(30%) to Zn-(52%) and to Cu-DAHP synthase (88%); 3) rate constant for product formation is higher in Mn- (130-200 s(-1)) than Cu- (55 s(-1)) and Zn- DAHP synthase (6.8 s(-1)); and 4) steady-state rate (rate constant for product release) is higher for the Mn(70 s(-1)) than for Cu- (5.6 s(-1)) and Zn- DAHP synthase (1.8 s(-1)). In addition, an examination of the reaction kinetics at lower pH reveals that for Cu- DAHP synthase, product release is no longer rate-limiting, whereas the Mn- and Zn- DAHP synthase show a slower rate of product formation, suggesting that the intermediate formation becomes rate-limiting in product formation. Also, a deuterium-isotope effect on the burst rate constant of product formation for Mn- DAHP synthase was observed at pH 6.0. This supports the hypothesis that the role of metal ion in E. coli DAHP synthase is to position the amino acids with the appropriate geometry required to coordinate and activate the water molecule.