Studies on 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe)from Escherichia coli K12. 2. Kinetic properties.
1. Co2+ is not a cofactor for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe). 2. The following analogues of phosphoenolpyruvate were tested as inhibitors of 3-deoxy-D-arabinoheptolosonate-7-phosphate synthetase(phe): pyruvate, lactate, glycerate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-methylphosphoenolpyruvate, 3-ethylphosphoenolpyruvate and 3,3-demethylphosphoenolpyruvate. The rusults obtained indicate that the binding of phosphoenolpyruvate to the enzyme requires a phosphoryl group on the C-2 position of the substrate and one free hydrogen atom at the C-3 position. 3. The dead-end inhibition pattern observed with the substrate analogue 2-phosphoglycerate when either phosphoenolpyruvate or erythrose 4-phosphate was the variable substrate is inconsistent with a ping-pong mechanism and indicates that the reaction mechanism for this enzyme must be sequential. The following kinetic constants were determined:Km for phosphoenolpyruvate, 0.08 +/- 0.04 mM; Km for erythrose 4-phosphate, 0.9 +/- 0.3 mM; K is for competitive inhibition by 2-phosphoglycerate with respect to phosphoenolpyruvate, 1.0 +/- 0.1 mM. 4. The enzyme was observed to have a bell-shaped pH PROFILE WITH A PH OPTIMUM OF 7.0. The effects of pH ON V and V/(Km for phosphoenolpyruvate) indicated that an ionizing group of pKa 8.0-8.1 is involved in the catalytic activity of the enzyme. The pKa of this group is unaffected by the binding of phosphoenolpyruvate.
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000378:metabolism*; D000446:Aldehyde-Lyases / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D003035:Cobalt / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005994:Glycerophosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D010649:Phenylalanine / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000031:analogs & derivatives / Q000494:pharmacology; D011485:Protein Binding; D013329:Structure-Activity Relationship
D005994:Glycerophosphates; D003035:Cobalt; D010649:Phenylalanine; D010728:Phosphoenolpyruvate; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D000446:Aldehyde-Lyases