LIMITED PROTEOLYSIS OF YEAST PHOSPHOFRUCTOKINASE - SEQUENCE LOCATIONSOF CLEAVAGE SITES CREATED BY THE ACTIONS OF DIFFERENT PROTEINASES

Purified phosphofructokinase 1 from baker's yeast (Saccharomyces cerev isiae) was subjected to proteolysis by thermolysin, endoproteinase lys -C, trypsin and chymotrypsin under defined solvent conditions. In the absence of substrates and allosteric effectors, the catalytic activity of phosphofructokinase rapidly disappeared in the presence of each pr oteolytic enzyme. The presence of a saturating concentration of ATP pr otected phosphofructokinase activity from proteolytic inactivation whi le the collective presence of fructose 6-phosphate, AMP and fructose 2 ,6-bisphosphate provided transient activation during proteolysis. Chan ges in the quaternary structure of phosphofructokinase resulting from proteolysis were estimated by high performance size exclusion chromato graphy while changes in the primary sequence of the individual alpha a nd beta polypeptide chains were estimated by polyacrylamide-gel electr ophoresis in sodium dodecylsulfate. The site(s) of proteolytic cleavag e were identified by N-terminal sequence analysis of resolved electrop horetic components. The presence of ATP protects phosphofructokinase f rom thermolysin proteolysis, while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate restricts proteolysis to one site in each polypeptide chain involving the peptide bonds prec eding Leu199 in the alpha chain and Leu192 in the beta chain. The trun cated phosphofructokinase retains its octameric structure. The presenc e of ATP largely restricts endoproteinase lys-C proteolysis to a singl e site in the alpha chain involving the peptide bond preceding Val914. This cleavage results in the dissociation of the octameric form of ph osphofructokinase into two tetramers. The presence of ATP restricts bo th trypsin and chymotrypsin proteolysis to the N-terminal and C-termin al regions described above, resulting in the preferential stabilizatio n of the tetrameric form of phosphofructokinase. It would appear that the first 200 and last 80 residues which are unique to the sequence of the yeast phosphosphofructokinase are not directly involved in cataly sis or its allosteric regulation. However, the last 80 residues of the alpha polypeptide chain do appear to stabilize an octameric structure which is unique to yeast phosphofructokinase.