Tryptophan Synthase a ! Subunit Glutamic Acid 49 Is Essential for Activity

We have obtained a complete set of 20 variants of the a 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 a subunits have been newly constructed by sitedirected mutagenesis using oligonucleotides. Six mutants were available from previous studies. We find that the wild type and all of the mutant a subunits form a& complexes with the 82 subunit of tryptophan synthase with similar association constants and similarly stimulate the activity of the D2 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 a subunit which significantly interferes with normal subunit interaction. However, the 19 mutant a d z complexes are completely devoid of activity in reactions normally catalyzed by the active site of the a subunit. This is the first time that these several activities have been measured with a series of highly purified a 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 a activity is especially significant and is strong evidence that glutamic acid 49 is an essential catalytic base in the reaction catalyzed by the a 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 a& complex of tryptophan synthase from Salmonella typhimurium.