Abstract
This chapter describes the assay, purification, and properties of anthranilate synthase. This enzyme catalyzes the formation of anthranilate from either chorismate and glutamine or chorismate and ammonia. It exists in E. coli and other organisms as an aggregate of two proteins: anthranilate synthase component I and phosphoribosyl (PR) transferase. The anthranilate synthase enzyme utilizes both glutamine and ammonia as amino donors. The formation of anthranilic acid is measured either colorimetrically using a modified Bratton and Marshall color reaction or followed fluorimetrically in an Aminco Bowman spectrofluorometer. The purified enzyme is stable for several months if kept in 0.1 M potassium phosphate buffer A containing 0.1 M sucrose at -70°. It loses activity on freezing and thawing, and this loss is more pronounced with glutamine as the amino donor than with NH 4 Cl. The pH optimum is 7.4 and 7.6 for glutamine as the amino donor and 8.7 for NH 4 CI as the amino donor. p -chloromercuribenzene sulfonic acid completely inhibits the enzymatic activity with both NH 3 and glutamine as amino donors. This inhibition can be reversed by thiol reagents.
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