Role of tryptophan 248 in the active site of tryptophanase from escherichia coli
Kawata, Y.; Tsujimoto, N.; Tani, S.; Mizobata, T.; Tokushige, M.
Fukui, T , Et Al (Ed ) International Union Of Biochemistry Symposium, 199 Enzymes Dependent on Pyridoxal Phosphate And Other Carbonyl Compounds As Cofactors; 8th International Symposium on Vitamin B6 And Carbonyl Catalysis, Osaka, Japan, October 15-19, 1990 Xviii+656p Pergamon Press: Oxford, England, Uk; New York, New York, Usa Illus 291-292
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.