The alpha subunit of tryptophan synthase. Evidence that aspartic acid 60 is a catalytic residue and that the double alteration of residues 175 and 211 in a second-site revertant restores the proper geometry of the substrate binding site

• Published in: The Journal of biological chemistry Vol. 264; no. 11; pp. 6288 - 6296
• Main Authors: NAGATA, S, HYDE, C. C, MILES, E. W
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15-04-1989
• Subjects: Analytical, structural and metabolic biochemistry; Aspartic Acid; Binding Sites; Biological and medical sciences; Biotechnology; Cloning, Molecular; DNA Mutational Analysis; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Genetic technics; Glycerophosphates - pharmacology; Indoles - pharmacology; Ligands; Lyases; Methods. Procedures. Technologies; Models, Molecular; Mutant screening; Prokaryotes; Protein Conformation; Recombinant Proteins; Salmonella typhimurium - enzymology; Substrate Specificity; tryptophan synthase; Tryptophan Synthase - genetics; Tryptophan Synthase - physiology; Enzyme; Active site; Tryptophan synthase; Site directed mutagenesis; Bacteria; Subunit; Escherichieae; Salmonella typhimurium; Enterobacteriaceae
• ISSN: 0021-9258; 1083-351X

Our studies, which are aimed at understanding the catalytic mechanism of the alpha subunit of tryptophan synthase from Salmonella
typhimurium, use site-directed mutagenesis to explore the functional roles of aspartic acid 60, tyrosine 175, and glycine
211. These residues are located close to the substrate binding site of the alpha subunit in the three-dimensional structure
of the tryptophan synthase alpha 2 beta 2 complex. Our finding that replacement of aspartic acid 60 by asparagine, alanine,
or tyrosine results in complete loss of activity in the reaction catalyzed by the alpha subunit supports a catalytic role
for aspartic acid 60. Since the mutant form with glutamic acid at position 60 has partial activity, glutamic acid 60 may serve
as an alternative catalytic base. The mutant form in which tyrosine 175 is replaced by phenylalanine has substantial activity;
thus the phenolic hydroxyl of tyrosine 175 is not essential for catalysis or substrate binding. Yanofsky and colleagues have
identified many missense mutant forms of the alpha subunit of tryptophan synthase from Escherichia coli. Two of these inactive
mutant forms had either tyrosine 175 replaced by cysteine or glycine 211 replaced by glutamic acid. Surprisingly, a second-site
revertant which contained both of these amino acid changes was partially active. These results indicated that the second mutation
must compensate in some way for the first. We now extend the studies of the effects of specific amino acid replacements at
positions 175 and 211 by two techniques: 1) characterization of several mutant forms of the alpha subunit from S. typhimurium
prepared by site-directed mutagenesis and 2) computer graphics modeling of the substrate binding site of the alpha subunit
using the x-ray coordinates of the wild type alpha 2 beta 2 complex from S. typhimurium. We conclude that the restoration
of alpha subunit activity in the doubly altered second-site revertant results from restoration of the proper geometry of the
substrate binding site.