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

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Abstract
Co2+ is not a cofactor for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) [EC 4.1.2.15]. The following analogs of phosphoenolpyruvate were tested as inhibitors of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe): pyruvate, lactate, glycerate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-methylphosphoenolpyruvate, 3-ethylphosphoenolpyruvate and 3,3-dimethylphosphoenolpyruvate. The results obtained indicate that the binding of phosphoenolpyruvate to the enzyme requires a phosphoryl group on the C-2 position of the substrate and 1 free H atom at the C-3 position. The dead-end inhibition pattern observed with the substrate analog 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. Km for phosphoenolpyruvate was 0.08 .+-. 0.04 mM, Km for erythrose 4-phosphate was 0.9 .+-. 0.3 mM and Ki for competitive inhibition by 2-phosphoglycerate with respect to phosphoenolpyruvate was 1.0 .+-. 0.1 mM. The enzyme had a bell-shaped pH profile with a pH optimum of 7.0. The effects of pH on V [velocity] 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.

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