title: Role of tryptophan 248 in the active site of tryptophanase from Escherichia coli.
abstract: Tryptophan 248, located in the active site of tryptophanase from Escherichia coli, has been replaced with phenylalanine by site-directed mutagenesis. Judging from CD and fluorescence spectra, the global structure of the mutant enzyme was found to be the same as that of the wild-type enzyme. The binding affinity of the mutant enzyme for the coenzyme pyridoxal 5'-phosphate (PLP) was reduced tenfold compared to the wild-type enzyme. Kinetic analyses under PLP-saturated conditions indicated that the Km values of the mutant enzyme for substrates are the same as those of wild-type enzyme but the kcat values are decreased to about 85%, which accounts for the overall activity decrease. These findings suggest that tryptophan 248 interacts closely with PLP and plays an important role in the catalytic reaction.
authors: Department of Chemistry, Faculty of Science, Kyoto University, Japan.
authors: Y
authors: Y
authors: Kawata
authors: N
authors: N
authors: Tsujimoto
authors: S
authors: S
authors: Tani
authors: T
authors: T
authors: Mizobata
authors: M
authors: M
authors: Tokushige
mesh_terms: D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014368:Tryptophanase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D010649:Phenylalanine; D011732:Pyridoxal Phosphate; D014364:Tryptophan; D014368:Tryptophanase