Importance of residues 2-9 in the immunoreactivity, subunit interactions, and activity of the beta 2 subunit of Escherichia coli tryptophan synthase.
The epitope recognized by a monoclonal antibody (mAb19) directed against the beta 2 subunit of Escherichia coli tryptophan synthase was found to be carried by residues 2-9 of the beta chain. The affinities of mAb19 for peptides of different lengths containing the 2-9 sequence were close to 0.6 x 10(9) M-1, the affinity of mAb19 for native beta 2. In view of these results, a model is proposed to account for the kinetics of appearance of the epitope during in vitro renaturation of beta 2 (Murry-Brelier, A., and Goldberg, M.E. (1988) Biochemistry 27, 7633-7640). A mutant producing beta chains lacking residues 1-9 (beta delta 1-9) was prepared. The beta delta 1-9 protein was able to fold into a heat stable homodimer resembling wild type beta 2. Isolated beta delta 1-9 had no detectable enzymatic activity. It could bind alpha chains extremely weakly and be slightly activated. In the presence of the 1-9 peptide, the beta delta 1-9 protein could bind alpha 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 beta chain appears strongly involved in the alpha-beta interactions and in the enzymatic activity.
Navon
A
A
Department of Life Sciences, Bar Ilan University, Ramat Gan, Israel.
Schulze
A J
AJ
Guillou
Y
Y
Zylinski
C A
CA
Baleux
F
F
Expert-Bezançon
N
N
Friguet
B
B
Djavadi-Ohaniance
L
L
Goldberg
M E
ME
D000911:Antibodies, Monoclonal / Q000276:immunology; D002942:Circular Dichroism; D003062:Codon; D004795:Enzyme Stability; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet; D014367:Tryptophan Synthase / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003062:Codon; D000939:Epitopes; D010446:Peptide Fragments; D014367:Tryptophan Synthase