LIMITED PROTEOLYSIS OF SACCHAROMYCES-CEREVISIAE PHOSPHOENOLPYRUVATE CARBOXYKINASE

Incubation of Saccharomyces cerevisiae phosphoenolpyruvate carboxykina se with trypsin under native conditions cases a time-dependent loss of activity and the production of protein fragments. Cleavage sites dete rmined by sodium dodecyl sulfate polyacrylamide gel electrophoresis an d sequence analyses identified protease-sensitive peptide bonds betwee n amino acid residues at positions 9-10 and 76-77. Additional fragment ation sites were also detected in a region approximately 70-80 amino a cids before the carboxyl end of the protein. These results suggest tha t the enzyme is formed by a central compact domain comprising more tha n two thirds of the whole protein structure. From proteolysis experime nts carried out in the presence of substrates, it could be inferred th at CO2 binding specifically protects position 76-77 from trypsin actio n. Intrinsic fluorescence measurements demonstrated that CO2 binding i nduces a protein conformational change, and a dissociation constant fo r the enzyme CO2 complex of 8.24 +/- 0.6 mM was determined