Insights into the mechanism of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (Phe) from Escherichia coli using a transient kinetic analysis.
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.
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, USA.
Cristina
C
Furdui
Lily
L
Zhou
Ronald W
RW
Woodard
Karen S
KS
Anderson
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002712:Chlorides / Q000494:pharmacology; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D017967:Zinc Compounds; C016837:zinc chloride; D003903:Deuterium; D010723:3-Deoxy-7-Phosphoheptulonate Synthase