title: Regulation of phenylalanine biosynthesis. Studies on the mechanism of phenylalanine binding and feedback inhibition in the Escherichia coli P-protein.
abstract: Isothermal titration calorimetry (ITC) and site-directed mutagenesis were used to study the interaction of Phe with (a) the Escherichia coli P-protein, a bifunctional chorismate mutase/prephenate dehydratase that is feedback inhibited by Phe, (b) PDT32, a 32 kDa P-protein fragment (residues 101-386) containing the prephenate dehydratase and regulatory domains, and (c) R12, a C-terminal 12 kDa P-protein fragment (residues 286-386) containing the regulatory domain. DeltaH(total) values for PDT32, which included the heats of Phe binding, conformational change, and dimerization, established that in developing a mechanism for end product feedback inhibition, the P-protein has evolved a ligand recognition domain that exhibits Phe-binding enthalpies comparable to those reported for other full-fledged amino acid receptor proteins. Sequence alignments of R12 with other Phe-binding enzymes identified two highly conserved regions, GALV (residues 309-312) and ESRP (residues 329-332). Site-directed mutagenesis and ITC established that changes in the GALV and ESRP regions affected Phe binding and feedback inhibition to different extents. Mutagenesis further showed that C374 was essential for feedback inhibition, but not for Phe binding, while W338 was involved in Phe binding, but not in the Phe-induced conformational change required for feedback inhibition.
authors.affiliation: Baker Laboratory, Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
authors.forename: G
authors.initials: G
authors.lastname: Pohnert
authors.forename: S
authors.initials: S
authors.lastname: Zhang
authors.forename: A
authors.initials: A
authors.lastname: Husain
authors.forename: D B
authors.initials: DB
authors.lastname: Wilson
authors.forename: B
authors.initials: B
authors.lastname: Ganem
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002151:Calorimetry; D002826:Chorismate Mutase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005246:Feedback; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D011302:Prephenate Dehydratase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C071216:P-protein, E coli; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010649:Phenylalanine; D011302:Prephenate Dehydratase; D002826:Chorismate Mutase