Regulation of aromatic amino acid biosynthesis in Bacillus subtilis 168. I. Evidence for and characterization of a trifunctional enzyme complex.

A bifunctional enzyme, consisting of 3-deoxy-n-arabinoheptulosonate 7-phosphate synthetase and chorismate mutase, has been purified to homogeneity from Bacillus subtilis. Ultracentrifugation revealed a single symmetrical peak of protein. A single protein band also was observed when the homogeneous preparation was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at pH 7.0. These data suggest that the protein consists of identical subunits. Following treatment of the purified protein with dimethyl suberimidate, four protein bands could be clearly distinguished upon electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. These four bands correspond to the monomer, dimer, trimer, and tetramer forms of the enzyme, as calculated from their molecular weights. The molecular weight of the monomer is about 38,500 as determined by its mobility following electrophoresis on polyacrylamide in the presence of sodium dodecyl sulfate. This number corresponds closely to the minimum chemical molecular weight as calculated from the amino acid composition. The enzyme can exist as a tetramer of molecular weight 160,000 as determined by superfine Sephadex G-200 thin layer chromatography. Metal ions and chemical inhibitors affect the two enzyme activities quite differently, Cd+2 stimulates 3-deoxy-narabinoheptulosonate 7-phosphate synthetase without any effect on chorismate mutase, while p-hydroxymercuribenzoate inhibits 3-deoxy-n-arabinoheptulosonate 7-phosphate synthetase but not chorismate mutase.