Purification and Properties of Bifunctional 3-Deoxy-D-arabino-Heptulosonate 7-Phosphate Synthetase-Chorismate Mutase Component A from Brevibacterium flavum

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Abstract
3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase of Brevibacterium flavum, which is the first enzyme in the aromatic amino acid biosynthetic pathway, was purified 540-fold to a disc-gel-electrophoretically homogeneous state. During the purification processes, component A of chorismate mutase, a common enzyme for phenylalanine and tyrosine biosynthesis, was purified together with DAHP synthetase in a constant ratio. It was concluded that DAHP synthetase and chorismate mutase component A form a bifunctional enzyme. The molecular weight of bifunctional DAHP synthetase-chorismate mutase component A was estimated to be 250,000 by gel-filtration. On SDS-polyacrylamide-gel electrophoresis, a single protein band was obtained and the molecular weight was estimated to be 55,000, suggesting that DAHP synthetase-chorismate mutase component A is an oligomer of identical subunits. 5, 5'-Dithio-bis(2-nitrobenzoic acid) (DTNB) reacted with 3 mol of sulfhydryl groups per mol of the subunit. One of the sulfhydryl groups was essential for the DAHP synthetase activity, whereas the chorismate mutase activity was not affected by DTNB at all. The optimum pH of the DAHP synthetase reaction was 6.5 with the purified preparation. The DAHP synthetase activity was stimulated 30% by chorismate mutase component B but not by chorismate, the substrate of chorismate mutase. Double-reciprocal plots of the reaction rate against erythrose-4-phosphate concentration gave straight lines with Km of 0.4 mM for erythrose-4-phosphate. Plots of the reaction rate against phosphoenolpyruvate concentration curved upwards. The Hill coefficient and S0.5 for phosphoenolpyruvate were estimated to be 4.1 and 0.4 mM, respectively. Inhibition by inorganic phosphate, one of the products, was noncompetitive for erythrose-4-phosphate and was not competitive for phosphoenol-pyruvate. Phenylalanine and tyrosine were competitive inhibitors for erythrose-4-phosphate (apparent K1, 0.066 mM) and mixed-type inhibitors for phosphoenolpyruvate. Tryptophan did not affect DAHP synthetase activity at all.

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