alpha-chymotrypsin and fructose-2-6-diphosphate

Purified phosphofructokinase 1 from baker's yeast (Saccharomyces cerevisiae) 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 proteolytic enzyme. The presence of a saturating concentration of ATP protected phosphofructokinase activity from proteolytic inactivation while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate provided transient activation during proteolysis. Changes in the quaternary structure of phosphofructokinase resulting from proteolysis were estimated by high performance size exclusion chromatography while changes in the primary sequence of the individual alpha and beta polypeptide chains were estimated by polyacrylamide-gel electrophoresis in sodium dodecylsulfate. The site(s) of proteolytic cleavage were identified by N-terminal sequence analysis of resolved electrophoretic components. The presence of ATP protects phosphofructokinase from 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 preceding Leu199 in the alpha chain and Leu192 in the beta chain. The truncated phosphofructokinase retains its octameric structure. The presence of ATP largely restricts endoproteinase lys-C proteolysis to a single site in the alpha chain involving the peptide bond preceding Val914. This cleavage results in the dissociation of the octameric form of phosphofructokinase into two tetramers. The presence of ATP restricts both trypsin and chymotrypsin proteolysis to the N-terminal and C-terminal regions described above, resulting in the preferential stabilization 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 phosphofructokinase are not directly involved in catalysis 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.

Topics: Adenosine Monophosphate; Adenosine Triphosphate; Amino Acid Sequence; Chromatography, High Pressure Liquid; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Fructosediphosphates; Fructosephosphates; Metalloendopeptidases; Molecular Sequence Data; Molecular Weight; Phosphofructokinase-1; Protein Conformation; Saccharomyces cerevisiae; Thermolysin

Phosphofructokinase from rabbit brain consists of hybrids of the A, B, and C isozymes. Phosphofructokinase C was isolated from a purified mixture of such hybrids in a 2-step procedure. In the first step, phosphofructokinase B was removed by chromatography on DEAE-Sephadex. In the second step, subunits of phosphofructokinases A and C were separated by dissociation at pH 5.0 followed by chromatography on carboxymethylcellulose. The separated isozymes were then reassociated by neutralization. Phosphofructokinase C was structurally distinct from phosphofructokinases A (obtained from muscle or brain) and B (obtained from liver) as shown by one-dimensional chymotryptic and staphylococcal V8 protease fingerprints of all three isozymes. In addition, phosphofructokinase C cross-reacted weakly or not at all with antisera raised against phosphofructokinase B or phosphofructokinase A. Phosphofructokinase C was also kinetically distinct from the A and B isozymes. The C isozyme was more sensitive than the A isozyme but less sensitive than the B isozyme to inhibition by ATP, was less sensitive than the A isozyme but more sensitive than the B isozyme to inhibition by citrate, and was less sensitive than either of the other two isozymes to activation by inorganic phosphate, AMP, and fructose 2,6-bisphosphate. The self-association properties of phosphofructokinase C differed from those of the A and B isozymes in that at pH 8.0, the C isozyme did not form oligomers larger than a tetramer under conditions where the other two isozymes did. Thus the properties of phosphofructokinase C are in general quite distinct from those of the other two phosphofructokinase isozymes.

Topics: Adenosine Monophosphate; Animals; Brain; Chromatography, Ion Exchange; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Enzyme Activation; Fructosediphosphates; Hydrogen-Ion Concentration; Isoenzymes; Kinetics; Muscles; Phosphates; Phosphofructokinase-1; Rabbits; Serine Endopeptidases