Role of tryptophan 248 in the active site of tryptophanase from Escherichia coli

Kawata, Y.; Tsujimoto, N.; Tani, S.; Mizobata, T.; Tokushige, M.

Biochemical and Biophysical Research Communications 173(2): 756-762

1990


ISSN/ISBN: 0006-291X
PMID: 2260981
DOI: 10.1016/s0006-291x(05)80100-7
Accession: 041291694

Download citation:  
Text
  |  
BibTeX
  |  
RIS

Article/Abstract emailed within 0-6 h
Payments are secure & encrypted
Powered by Stripe
Powered by PayPal

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.