Studies on 3‐Deoxy‐ d ‐ arabino heptulosonate‐7‐phosphate Synthetase(phe) from Escherichia coli K12

1 Co 2+ is not a cofactor for 3‐deoxy‐ d ‐ arabino heptulosonate‐7‐phosphate synthetase(phe). 2 The following analogues of phosphoenolpyruvate were tested as inhibitors of 3deoxy‐ d ‐arabinoheptolosonate‐7‐phosphate synthetase(phe): pyruvate, lactate, glycerate, 2‐phosphoglycerate, 3‐methylphospho enol pyruvate, 3‐ethylphosphoenolpyruvate and 3,3‐dime‐thylphoso enol pyruvate. The results obtained indicate that the binding of phospho enol pyruvate 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: K m for phosphoenolpyruvate, 0.08 ± 0.04 mM: K m for erythrose 4‐phosphate, 0.9 ± 0.3 mM; Kis for competitive inhibition by 2‐phosphoglycerate with respect to phosphoenolpyruvate, 1.0 ± 0.1 mM. 4 The enzvmc was observed to have a bell‐shaped pH profile with a pH optimum of 7.0. The effects of pH on V and V/( K m for phosphoenolpyruvate) indicated that an ionizing group of pK a .8.0–8.1 is involved in the catalytic activity of the enzyme. The p K a of this group is unallected by the binding of phosphoenolpyruvate.