title: Site-directed mutagenesis of monofunctional chorismate mutase engineered from the E. coli P-protein.
abstract: Analysis of the active-site residues of a fully functional chorismate mutase representing the N-terminal 113 amino acids of the Escherichia coli P-protein suggests that Lys39 and Gln88 play critical roles in catalyzing the rearrangement of chorismate to prephenate. Five site-directed mutants at these positions have been constructed in which Lys39 was replaced with Arg, Asn, and Gln, and Gln88 was replaced with Arg and Glu. Although the Gln88Arg plasmid failed to produce detectable cross-reacting proteins in E. coli, the other four plasmids were expressed, and the mutant proteins purified to homogeneity. Their structures were similar to wild type enzyme, as indicated by circular dichroism spectra, with Lys39Gln showing a small deviation. In accordance with predictions, all mutations result in major loss of catalytic activity at pH 7.8. However, activity of the Gln88Glu mutant at pH 4.5 exceeded wild-type EcCM. Implications for the mechanism of mutase catalysis are discussed.
authors.affiliation: Section of Biochemistry, Molecular and Cellular Biology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: S
authors.initials: S
authors.lastname: Zhang
authors.forename: P
authors.initials: P
authors.lastname: Kongsaeree
authors.forename: J
authors.initials: J
authors.lastname: Clardy
authors.forename: D B
authors.initials: DB
authors.lastname: Wilson
authors.forename: B
authors.initials: B
authors.lastname: Ganem
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D002826:Chorismate Mutase / Q000235:genetics*; D002942:Circular Dichroism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D007700:Kinetics; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D011302:Prephenate Dehydratase / Q000235:genetics*; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C071216:P-protein, E coli; D011302:Prephenate Dehydratase; D002826:Chorismate Mutase