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

Abstract

1. Co2+ is not a cofactor for 3-deoxy-~-arubi~zoheptulosonate-7-phosphate synthetase(phe). 2. The following analogues of phosphoenolpyruvate were tested as inhibitors of 3-deoxy-~-arabinoheptolosonate-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 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, for phosphoenolpyruvate, 0.08 0.04 mM; K , for erythrose 4-phosphate, 0.9 0.3 mM; Ki, for competitive inhibition by 2-phosphoglycerate with respect to phosphoenolpyruvate, 1 .O 4. The enzyme was observed to have a bell-shaped pH profile with a pH optimum of 7.0.The effects of pH on Y and V/(Km for phosphoenofpyruvate) indicated that an ionizing group of pK, 8.0-8.1 is involved in the catalytic activity of the enzyme. The pK, of this group is unaffected by the binding of phosphoenolpyruvate. mM.Previous characterizations of the kinetic properties of 3-deoxy-~-arahinoheptulosonate-7-phosphate synthetase(phe) from Escherichia coli have produced conflicting reports concerning the details of its inhibition by phenylalanine and the possible role of Co2+ as a cofactor for the enzyme. Staub and Denes [l] observed that the kinetics of phenylalanine inhibition were essentially hyperbolic (Hill coefficient, n' = 1.2) and that the addition of Co2+ enhanced enzyme activity markedly, while Simpson et al.[2] reported sigmoidal inhibition kinetics for phenylalanine (n' = 2.0) and little, if any, effect of Co2+ on enzyme activity.The original reports also suggested that the enzyme mechanism was ping pong, with phosphoenolpyruvate being the first substrate to react with the enzyme [3,4] ; however, DeLeo et al. reported more recently that 3-deoxy-~-arubinoheptulosonate-7-phosphate synthetase(tyr) from Salmonella typhimurium follows an ___ Enzymes. 2-Deoxy-~-arabinoheptulosonate-7-phosphate synthetase(phe) (EC 4.1.2.15); pyruvate kinase (EC 2.7.1.40). ordered sequential mechanism [ 5 ] . It seems unlikely that these enzymes would have different reaction mechanisms although they are different isoenzymes from different genera.With the recent purification to near homogeneity of the 3-deoxy-~-arabinoheptulosonate-7-phosphate synthetase(phe) from E. coli [6] we decided to undertake further kinetic studies with this enzyme, particularly since the strain used in this purification was different to that reported on previously [ 2 ] . These studies included a re-examination of the effects of phenylalanine and Co2 + ...

Aromatic amino acid biosynthesis in the yeast Saccharomyces cerevisiae: a model system for the regulation of a eukaryotic biosynthetic pathway.

“…The enzyme can be inactivated by EDTA in a reaction which can be reversed by the addition of several bivalent metal ions. Similar results have been found for the phenylalanine-inhibitable E. coli enzyme (123,175,179).…”

“…The kinetic data of the phenylalanine-inhibitable yeast DAHP synthase (summarized in Table 5), with a calculated rate constant of 10 s-1 (156), suggest a sequential reaction mechanism similar to that proposed for the tyrosine-inhibitable E. coli DAHP synthase (171). The apparent Michaelis constant of the enzyme is 0.018 mM for PEP, which is similar to the values obtained for the two available DAHP synthase isoenzymes from E. coli and the tryptophan-sensitive enzyme from N. crassa (124,145,171,175). For E4P the Michaelis constant is 0.13 mM, and the reported values for the other described enzymes range between 0.0027 and 0.9 mM.…”

“…Gel filtration indicates that the active enzyme is a monomer with a molecular mass of 42 kDa, which corresponds to the calculated molecular mass deduced from the previously determined primary sequence (154). A comparison with the three described DAHP synthases from E. coli reveals striking differences in the quaternary structure: the phenylalanine-inhibitable enzyme is a tetramer, whereas the tyrosineinhibitable enzyme is a dimer (124,171,175), although there is 70% sequence similarity between the enzymes (52,174). The tryptophan-inhibitable DAHP synthase has been described as a tetramer (145).…”