Tryptophan synthase alpha subunit glutamic acid 49 is essential for activity. Studies with 19 mutants at position 49.
We have obtained a complete set of 20 variants of the alpha subunit of tryptophan synthase of Escherichia coli at position 49 in order to extend our previous studies on the effects of single amino acid replacements at position 49 on structure and function. Thirteen mutant alpha subunits have been newly constructed by site-directed mutagenesis using oligonucleotides. Six mutants were available from previous studies. We find that the wild type and all of the mutant alpha subunits form alpha 2 beta 2 complexes with the beta 2 subunit of tryptophan synthase with similar association constants and similarly stimulate the activity of the beta 2 subunit in the synthesis of L-tryptophan from L-serine and indole. Thus none of the changes at position 49 produces a change in the conformation of the alpha subunit which significantly interferes with normal subunit interaction. However, the 19 mutant alpha 2 beta 2 complexes are completely devoid of activity in reactions normally catalyzed by the active site of the alpha subunit. This is the first time that these several activities have been measured with a series of highly purified alpha subunits altered by mutation at a single site. Our finding that the mutant in which glutamic acid 49 is substituted by aspartic acid is totally devoid of alpha activity is especially significant and is strong evidence that glutamic acid 49 is an essential catalytic base in the reaction catalyzed by the alpha subunit. This result is consistent with the results of previous genetic studies, with evolutionary comparisons using sequence analysis, and with recent results from x-ray crystallography of the alpha 2 beta 2 complex of tryptophan synthase from Salmonella typhimurium.
D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005971:Glutamates; D018698:Glutamic Acid; D046911:Macromolecular Substances; D009154:Mutation; D013698:Templates, Genetic; D014367:Tryptophan Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D005971:Glutamates; D046911:Macromolecular Substances; D018698:Glutamic Acid; D014367:Tryptophan Synthase