Purification and phosphorylation of fructose-1,6-bisphosphatase from Kluyveromyces fragilis.
Fructose-1,6-bisphosphatase from the yeast Kluyveromyces fragilis was found to have an apparent Mr = 155,000 and to be composed of four Mr = 35,000 subunits. The extent and rate of phosphorylation of fructose-1,6-bisphosphatase (Fru-1,6-P2) by yeast cAMP-dependent protein kinase were dependent on fructose-1,6-bisphosphatase inhibitors, 5'-AMP and fructose 2,6-bisphosphate (Fru-2,6-P2). In the absence of inhibitor, the enzyme was slowly phosphorylated with a maximum incorporation of 1 mol of phosphate/mol of enzyme. The presence of both inhibitors greatly increased the phosphorylation rate with a maximum incorporation of 2 mol of phosphate/mol of enzyme. The presence of only one inhibitor led to an intermediate rate of phosphorylation with 2 mol of phosphate incorporated/mol of enzyme. There was no significant change in enzymatic activity after phosphorylation. The estimated sedimentation coefficient of fructose-1,6-bisphosphatase was lowered by 5'-AMP from 8.2 to 5.7 while Fru-2,6-P2 increased the S value to 8.5. The presence of either Fru-1,6-P2 or Fru-2,6-P2 prevented the 5'-AMP lowering of S value. The susceptibility of enzyme to partial tryptic digestion was not changed by the presence of 5'-AMP. The presence of both Fru-2,6-P2 and 5'-AMP led to the protection of Mr = 35,000 subunit from tryptic digestion while Fru-2,6-P2 alone led to a protection of an Mr = 30,000 peptide fragment. This peptide fragment did not contain the phosphorylation sites. Our results suggest that the rapid regulation of fructose-1,6-bisphosphatase following glucose addition is controlled mainly by enzyme inhibitors.
D001203:Ascomycota / Q000201:enzymology*; D006597:Fructose-Bisphosphatase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D004718:Saccharomycetales / Q000201:enzymology*; D014357:Trypsin
D010446:Peptide Fragments; D006597:Fructose-Bisphosphatase; D014357:Trypsin