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)

[1]  H. Brewer,et al.  The chemical ionization mass spectrometric analysis of phenylthiohydantoin and 2-anilino-5-thiazolinone amino acids obtained from the Edman degradation of proteins and peptides. , 1980, Analytical biochemistry.

[2]  Jeffrey W. Roberts,et al.  遺伝子の分子生物学 = Molecular biology of the gene , 1970 .

[3]  M. Goldberg,et al.  Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[4]  E. W. Miles,et al.  An active alpha'2beta2 derivative of tryptophean synthase formed by limited proteolysis. , 1978, The Journal of biological chemistry.

[5]  K. Tsujii,et al.  Free-boundary electrophoresis of sodium dodecyl sulfate-protein polypeptide complexes with special reference to SDS-polyacrylamide gel electrophoresis. , 1974, Journal of biochemistry.

[6]  I. Crawford,et al.  Assignment of the ends of the beta-chain of E. coli tryptophan synthase to the F1 and F2 domains. , 1978, Biochemical and biophysical research communications.

[7]  K. Kirschner,et al.  Kinetics of cooperative ligand binding to the apo beta 2 subunit of tryptophan synthase and its modulation by the alp ha subunit. , 1980, Biochemistry.

[8]  L. Flohé,et al.  The complete amino acid sequence of low molecular mass urokinase from human urine. , 1982, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[9]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[10]  E. W. Miles,et al.  An active proteolytic derivative of the alpha subunit of tryptophan synthase. Identification of the site of cleavage and characterization of the fragments. , 1979, Biochemistry.

[11]  T. Takagi,et al.  Interaction between the α1 chain of rat tail collagen and sodium dodecyl sulfate with reference to its behavior in sds-polyacrylamide gel electrophoresis , 1982 .

[12]  J. W. Little,et al.  Deletions within a hinge region of a specific DNA-binding protein. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[13]  R. Ambler.  [49] Carboxypeptidases A and B , 1967 .

[14]  C. A. Knight,et al.  Effect of charge on the determination of molecular weight of proteins by gel electrophoresis in SDS. , 1971, Biochemical and biophysical research communications.

[15]  I. Crawford,et al.  Studies on the association of beta-chain monomers of Escherichia coli tryptophan synthetase. , 1970, Biochemistry.

[16]  K. Tsujii,et al.  Binding isotherms of sodium dodecyl sulfate to protein polypeptides with special reference to SDS-polyacylamide gel electrophoresis. , 1975, Journal of biochemistry.

[17]  J. Richardson,et al.  The anatomy and taxonomy of protein structure. , 1981, Advances in protein chemistry.

[18]  K. Yutani,et al.  Guanidine hydrochloride induced unfolding of the alpha subunit of tryptophan synthase and of the two alpha proteolytic fragments: evidence for stepwise unfolding of the two alpha domains. , 1982, Biochemistry.

[19]  B. Frangione,et al.  Proteolytic enzymes from the mouse submaxillary gland. Specificity restricted to arginine residues. , 1977, Archives of biochemistry and biophysics.

[20]  D. Wetlaufer,et al.  Folding of protein fragments. , 1981, Advances in protein chemistry.

[21]  W. Rutter,et al.  Redesigning trypsin: alteration of substrate specificity. , 1985, Science.

[22]  P. Jekel,et al.  Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis. , 1983, Analytical biochemistry.

[23]  H. M. Lee,et al.  Does carboxypeptidase Y have intrinsic endopeptidase activity? , 1978, Biochemical and biophysical research communications.

[24]  E. W. Miles,et al.  Location of three active site residues in the NH2-terminal sequence of the beta 2 subunit tryptophan synthase from Escherichia coli. , 1980, Journal of Biological Chemistry.

[25]  D. Davies,et al.  Crystallization and preliminary X-ray crystallographic data of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. , 1985, Journal of Biological Chemistry.

[26]  C. Yanofsky,et al.  Nucleotide sequence of the trpB gene in Escherichia coli and Salmonella typhimurium. , 1980, Journal of molecular biology.

[27]  R. Hayashi.  [8] Car☐ypeptidase Y in sequence determination of peptides , 1977 .

[28]  I. Svendsen,et al.  Use of carboxypeptidase Y for carboxy-terminal sequence determination in proteins , 1977 .

[29]  M. Goldberg,et al.  Isolation and characterization of independently folding regions of the beta chain of Escherichia coli tryptophan synthetase. , 1977, Biochemistry.

[30]  H. Brewer,et al.  Automated Edman degradations: studies with a large sequencer cup and high-speed drive. , 1979, Analytical biochemistry.