PMID:9497350
| Citation |
Zhang, S, Pohnert, G, Kongsaeree, P, Wilson, DB, Clardy, J and Ganem, B (1998) Chorismate mutase-prephenate dehydratase from Escherichia coli. Study of catalytic and regulatory domains using genetically engineered proteins. J. Biol. Chem. 273:6248-53 |
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| Abstract |
The bifunctional P-protein, which plays a central role in Escherichia coli phenylalanine biosynthesis, contains two catalytic domains (chorismate mutase and prephenate dehydratase activities) as well as one R-domain (for feedback inhibition by phenylalanine). Six genes coding for P-protein domains or subdomains were constructed and successfully expressed. Proteins containing residues 1-285 and residues 1-300 retained full mutase and dehydratase activity, but exhibited no feedback inhibition. Proteins containing residues 101-386 and residues 101-300 retained full dehydratase activity, but lacked mutase activity. Fluorescence emission spectra and binding assays indicated that residues 286-386 were crucial for phenylalanine binding. The mutase (residues 1-109), dehydratase (residues 101-285), and regulatory (residues 286-386) activities were thus shown to reside in discrete domains of the P-protein. Both the mutase domain and the native P-protein formed dimers. Deletion of the mutase domain diminished phenylalanine binding to the regulatory 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 for chorismate. |
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| Keywords |
Allosteric Regulation; Bacterial Proteins/metabolism; Binding Sites; Chorismate Mutase/drug effects; Chorismate Mutase/genetics; Chorismate Mutase/metabolism; Cloning, Molecular; Dimerization; Escherichia coli/enzymology; Escherichia coli Proteins; Feedback; Hot Temperature; Multienzyme Complexes/drug effects; Multienzyme Complexes/genetics; Multienzyme Complexes/metabolism; Peptide Fragments/drug effects; Peptide Fragments/genetics; Peptide Fragments/metabolism; Phenylalanine/pharmacology; Prephenate Dehydratase/drug effects; Prephenate Dehydratase/genetics; Prephenate Dehydratase/metabolism; Protein Denaturation; Protein Engineering; Recombinant Proteins/drug effects; Recombinant Proteins/metabolism |
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