Purification and Properties of Anthranilate Synthase from Salmonella typhimurium

Abstract

Abstract The enzyme anthranilate synthase catalyzes the formation of anthranilic acid from chorismate and glutamine or chorismate and NH3 in the biosynthesis of tryptophan in bacteria and in yeast. A purification procedure involving the solubilization of this enzyme and several additional steps led to a preparation that is homogeneous as judged by gel filtrations, sedimentation pattern, and gel electrophoresis. The final yield was at least 25%. Several properties of the purified enzyme were studied. The molecular weight of the enzyme was found to be 137,000. The stoichiometry of the reaction with glutamine or ammonia as amino donor was determined. The pH optimum for glutamine was found to be 7.5 and for NH3 8.7. The Michaelis constant for chorismate is 3.3 x 10-6 m at pH 7.5, and the Michaelis constants for glutamine and NH3 are 5 and 8.5 x 10-4 m, respectively, at their individual pH optima. NH3 rather than NH4+ seems to be the active aminating species. A series of inhibitors was found which affected the activity of the enzyme either with glutamine or with ammonia or with both. The significance of these results is discussed in relation to the structure of the enzyme. Inhibition by the end product, tryptophan, was competitive with chorismate, and the kinetic behavior of the inhibition was allosteric in nature. No cooperative effects could be demonstrated with chorismate as substrate in the absence of the feedback inhibitor. The inhibition with tryptophan was complete.