title: Limited proteolysis of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.
abstract: Incubation of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with trypsin under native conditions cases a time-dependent loss of activity and the production of protein fragments. Cleavage sites determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and sequence analyses identified protease-sensitive peptide bonds between amino acid residues at positions 9-10 and 76-77. Additional fragmentation sites were also detected in a region approximately 70-80 amino acids before the carboxyl end of the protein. These results suggest that the enzyme is formed by a central compact domain comprising more than two thirds of the whole protein structure. From proteolysis experiments carried out in the presence of substrates, it could be inferred that CO2 binding specifically protects position 76-77 from trypsin action. Intrinsic fluorescence measurements demonstrated that CO2 binding induces a protein conformational change, and a dissociation constant for the enzyme CO2 complex of 8.2 +/- 0.6 mM was determined.
authors.affiliation: Departamento de Quimica, Facultad de Ciencia, Universidad de Santiago de Chile, Santiago.
authors.forename: L
authors.initials: L
authors.lastname: Herrera
authors.forename: M V
authors.initials: MV
authors.lastname: Encinas
authors.forename: A M
authors.initials: AM
authors.lastname: Jabalquinto
authors.forename: E
authors.initials: E
authors.lastname: Cardemil
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002245:Carbon Dioxide / Q000378:metabolism; D002918:Chymotrypsin; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014357:Trypsin
chemical_list: D010446:Peptide Fragments; D002245:Carbon Dioxide; D002918:Chymotrypsin; D014357:Trypsin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D010729:Phosphoenolpyruvate Carboxykinase (GTP)