Purification and characterization of an inducible aromatic amino acid-sensitive form of chorismate mutase from Solanum tuberosum L. tubers.
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 (S0.5 = 188 microM). However, a shift from sigmoidal to hyperbolic kinetics was observed when assays were performed at pH 8.5. Addition of 9 microM tryptophan to the assay resulted in maximum activation of the enzyme with a Ka of 1.2 microM. 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 Km for chorismate from 185 to 45 microM. Tryptophan (0.1 mM) completely protected CM from inhibition by phenylalanine (1.8 mM) and tyrosine (1.8 mM). However, in the absence of the activator, phenylalanine and tyrosine exhibited 50% inhibition at 0.80 and 0.68 mM concentrations, respectively. Both phenylalanine and tyrosine competitively inhibited CM activity with Ki values of 550 and 440 mM, respectively. Arogenate (1.0 mM) had no effect on CM activity in either the presence or absence of tryptophan. Analytical isoelectric focusing yielded an isoelectric point of 4.73.
Kuroki
G W
GW
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Conn
E E
EE
D000596:Amino Acids / Q000494:pharmacology*; D001667:Binding, Competitive; D002826:Chorismate Mutase / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007535:Isomerases / Q000302:isolation & purification*; D007700:Kinetics; D010649:Phenylalanine / Q000494:pharmacology; D010944:Plants / Q000201:enzymology*; D014364:Tryptophan / Q000494:pharmacology; D014443:Tyrosine / Q000494:pharmacology
D000596:Amino Acids; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D007535:Isomerases; D002826:Chorismate Mutase