Importance of residues 2-9 in the immunoreactivity, subunit interactions, and activity of the β2 subunit of Escherichia coli tryptophan synthase

Abstract

The epitope recognized by a monoclonal antibody (mAb19) directed against the β2 subunit of Escherichia coli tryptophan synthase was found to be carried by residues 2-9 of the β chain. The affinities of mAb19 for peptides of different lengths containing the 2-9 sequence were close to 0.6 x 109 M-1, the affinity of mAb19 for native β2. In view of these results, a model is proposed to account for the kinetics of appearance of the epitope during in vitro renaturation of β2 (Murry-Brelier, A., and Goldberg, M. E. (1988) Biochemistry 27, 7633-7640). A mutant producing β chains lacking residues 1- 9 (β(Δ1-9)) was prepared. The β(Δ1-9) protein was able to fold into a heat stable homodimer resembling wild type β2. Isolated β(Δ1-9) had no detectable enzymatic activity. It could bind a chains extremely weakly and be slightly activated. In the presence of the 1-9 peptide, the β(Δ1-9) protein could bind α chains much more strongly and generate a 50% active enzyme. Thus, although having little role in the overall folding and stability of the protein, the 1-9 sequence of the β chain appears strongly involved in the α-β interactions and in the enzymatic activity.