Purification and characterization of an inducible aromatic amino acid-sensitive form of chorismate mutase from Solanum tuberosum L. tubers

Abstract

An amino acid-sensitive form of chorismate mutase (CM) has been purified over 1000-fold from disks excised from tubers of Solanum tuberosum L. cv White Rose. Purification was accomplished by chromatography on Matrix Blue A followed by affinity chromatography with tryptophan as ligand. CM assays performed in the absence of tryptophan yielded pH-dependent sigmoidal kinetics. At pH 8.0, sigmoidal kinetics were observed with a Hill coefficient of 1.66 ( S 0.5 = 188 μM). However, a shift from sigmoidal to hyperbolic kinetics was observed when assays were performed at pH 8.5. Addition of 9 μ m tryptophan to the assay resulted in maximum activation of the enzyme with a K a of 1.2 μ m. When assayed in the presence of tryptophan, hyperbolic kinetics were observed over the pH range 6.0–8.0. Addition of tryptophan also decreased the K m for chorismate from 185 to 45 μ m. Tryptophan (0.1 m m) completely protected CM from inhibition by phenylalanine (1.8 m m) and tyrosine (1.8 m m). However, in the absence of the activator, phenylalanine and tyrosine exhibited 50% inhibition at 0.80 and 0.68 m m concentrations, respectively. Both phenylalanine and tyrosine competitively inhibited CM activity with K i values of 550 and 440 m m, respectively. Arogenate (1.0 m m) had no effect on CM activity in either the presence or absence of tryptophan. Analytical isoelectric focusing yielded an isoelectric point of 4.73.