CHORISMATE MUTASE-PREPHENATE DEHYDRATASE FROM ESCHERICHIA-COLI - STUDY OF CATALYTIC AND REGULATORY DOMAINS USING GENETICALLY-ENGINEERED PROTEINS

The bifunctional P-protein, which plays a central role in Escherichia coli phenylalanine biosynthesis, contains two catalytic domains (chori smate mutase and prephenate dehydratase activities) as well as one R-d omain (for feedback inhibition by phenylalanine). Six genes coding for P-protein domains or subdomains were constructed and successfully exp ressed. Proteins containing residues 1-285 and residues 1-300 retained full mutase and dehydratase activity, but exhibited no feedback inhib ition. Proteins containing residues 101-386 and residues 101-300 retai ned full dehydratase activity, but lacked mutase activity. Fluorescenc e emission spectra and binding assays indicated that residues 286-386 were crucial for phenylalanine binding. The mutase (residues 1-109), d ehydratase (residues 101-285), and regulatory (residues 286-386) activ ities were thus shown to reside in discrete domains of the P-protein. Both the mutase domain and the native P-protein formed dimers. Deletio n of the mutase domain diminished phenylalanine binding to the regulat ory site as well as prephenate binding to the dehydratase domain, both through cooperative effects. Besides eliminating feedback inhibition, removal of the R-domain decreased the affinity of chorismate mutase f or chorismate.