Site-directed mutagenesis of the beta subunit of tryptophan synthase from Salmonella typhimurium. Role of active site glutamic acid 350.
To investigate the functional role of glutamic acid 350 in the active site of the beta subunit of tryptophan synthase from Salmonella typhimurium, we have replaced this residue by glutamine or alanine by use of site-directed mutagenesis. The mutant alpha 2 beta 2 complexes were expressed, purified, crystallized, and characterized by spectroscopic and kinetic studies with several substrates. We find large alterations in the substrate and reaction specificity of each mutant form of the alpha 2 beta 2 complex. Since the two mutant enzymes are virtually inactive in reactions with L-serine but are active in reactions with beta-chloro-L-alanine, glutamic acid 350 may facilitate the beta-elimination of the weak hydroxyl leaving group of L-serine. The mutant alpha 2 beta 2 complexes are more active than the wild type enzyme in the beta-elimination reaction with beta-chloro-L-alanine. These enzymes are irreversibly inactivated by beta-chloro-L-alanine, whereas the wild type enzyme is not. These altered properties may result from a change in the conformation of the active site, from a change in the orientation of the coenzyme relative to active site residues, or from a change in the solvent accessibility of the active site. The alteration in the active site may enhance the release of amino acrylate from the Schiff base intermediate by hydrolysis or by transamination.
Kayastha
A M
AM
Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Sawa
Y
Y
Nagata
S
S
Miles
E W
EW
D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D005454:Fluorescence Polarization; D005971:Glutamates / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012486:Salmonella typhimurium / Q000201:enzymology*; D013057:Spectrum Analysis; D014367:Tryptophan Synthase / Q000235:genetics*
D000596:Amino Acids; D005971:Glutamates; D018698:Glutamic Acid; D014367:Tryptophan Synthase