Abstract
Escherichia coli phenylalanine-sensitive 3-deoxy-arabino-heptulosonate 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.
Highlights
The first committed step in the biosynthesis of shikimic acid, which leads to the synthesis of aromatic amino acids, is catalyzed by 3-deoxy-arabino-heptulosonate 7-phosphate (DAHP)1 synthase
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 is higher for the Mn(70 s؊1) than for Cu- (5.6 s؊1) and Zn-DAHP synthase (1.8 s؊1)
We have focused on the phenylalanine-sensitive DAHP synthase encoded by E. coli aroG gene
Summary
Throughout the paper we used DAHP synthase to refer to the isolated “as is” DAHP synthase and apoDAHP synthase for the EDTA-treated DAHP synthase (metal-free). Burst Experiment for the Zn-DAHP Synthase—ApoDAHP synthase (5 M), 5 M ZnCl2, and 15 M [1-14C]PEP were reacted with 500 M E4P in 50 mM BTP, pH 7.6 and pH 6.0. To follow the dependence of presteady-state burst experiments on DAHP synthase concentration, burst experiments were performed at three different enzyme concentrations: 3, 5, and 7.5 M. To examine the effect of pH, a burst experiment for the Cu-DAHP synthase was conducted at pH 6.5 For this experiment, 10 M apoDAHP synthase, 2 mM CuCl2, and 30 M [1-14C]PEP were reacted with 500 M E4P in 50 mM BTP, pH 6.5, followed by quenching with 0.3 M KOH at the desired time interval.
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