Chorismate Mutase-Prephenate Dehydratase: EVIDENCE FOR DISTINCT CATALYTIC AND REGULATORY SITES

Abstract

Abstract Chorismate mutase-prephenate dehydratase catalyzes the first two reactions specific for phenylalanine synthesis in Salmonella typhimurium. Both activities are subject to feedback inhibition by phenylalanine. Experiments with modifying reagents (bromopyruvate and p-mercuribenzoate), a product analogue (2,4-dihydroxybenzoate), and mutationally altered enzymes provide evidence for functionally distinct catalytic sites and a regulatory site distinct from the catalytic sites. The following evidence argues for functionally distinct sites for chorismate mutase and prephenate dehydratase activities. (a) Treatment with bromopyruvate or p-mercuribenzoate selectively inactivates prephenate dehydratase. (b) Prephenate dehydratase is selectively inhibited by 2,4-dihydroxybenzoate. (c) A mutant enzyme from a phenylalanine auxotroph is found to lack prephenate dehydratase activity while retaining chorismate mutase activity. A regulatory site for phenylalanine distinct from the catalytic sites is suggested by the following. (a) Treatment of chorismate mutase-prephenate dehydratase with bromopyruvate or p-mercuribenzoate desensitizes chorismate mutase to inhibition by phenylalanine. (b) A mutant chorismate mutase-prephenate dehydratase isolated from a 4-fluorophenylalanine-resistant strain is insensitive to inhibition by phenylalanine. It is suspected that this enzyme still binds phenylalanine because of its affinity for phenylalanine-substituted Sepharose. Other experiments, however, suggest some relationship between the catalytic site for prephenate dehydratase and a regulatory site for phenylalanine.