Identification of three sites of proteolytic cleavage in the hinge region between the two domains of the beta 2 subunit of tryptophan synthase of Escherichia coli or Salmonella typhimurium.
The beta 2 subunit of tryptophan synthase is composed of two independently folding domains connected by a hinge segment of the polypeptide that is particularly susceptible to limited proteolysis by trypsin [Högberg-Raibaud, A., & Goldberg, M. (1977) Biochemistry 16, 4014-4019]. Since tryptic cleavage in the hinge region inactivates the beta 2 subunit, the spatial relationship between the two domains is important for enzyme activity. However, it was not previously known whether inactivation results from cleavage of the chain or from the loss of internal fragment(s) subsequent to cleavage at two or more sites. We now report comparative studies of limited proteolysis by three proteinases: trypsin and endoproteinases Lys-C and Arg-C. Our key finding that endoproteinase Arg-C inactivates the beta 2 subunit by cleavage at a single site (Arg-275) demonstrates the important role of the hinge peptide for enzymatic activity. We have also identified the sites of cleavage and the time course of proteolysis by trypsin at Arg-275, Lys-283, and Lys-272 and by endoproteinase Lys-C at Lys-283 and Lys-272. Sodium dodecyl sulfate gel electrophoresis, Edman degradation, and carboxypeptidases B and Y have been used to identify the several fragments and peptides produced. Our finding that the beta 2 subunit and F1 fragments have a heterogeneous amino terminus (Met-1 or Thr-2) indicates that the amino-terminal methionine is incompletely removed during posttranslational modification. Our results show that Edman degradation can be effectively used with a protein of known sequence to analyze proteolytic digests that have at least four different amino-terminal sequences.(ABSTRACT TRUNCATED AT 250 WORDS)
D000596:Amino Acids / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology*; D014367:Tryptophan Synthase
D000596:Amino Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010447:Peptide Hydrolases; D014367:Tryptophan Synthase