Modification of Yeast Fructose-1,6-Bisphosphatase

Abstract

Phosphorylation of purified FBPase a can be demonstrated in vitro by incubation with purified cyclic AMP-dependent protein kinase from yeast in the presence of ATP, Mg2+, and cyclic AMP. The final loss of catalytic activity is about 60%, a value very similar to that found in intact cells 1–3 minutes after addition of glucose to starved yeast. The in vitro phosphorylated enzyme shows characteristic kinetic properties similar to the enzyme assayed in a crude extract from glucose-treated yeast and to FBPase-P b purified from such an extract. Incubation of FBPase a with a lysate from yeast vacuoles, containing the bulk of the proteolytic activity of yeast, shows inactivation kinetics similar to those observed after incubation of purified FBPase a with cyclic AMP-dependent protein kinase. Chymostatin and the endogenous proteinase inhibitor IB from yeast, both known as specific inhibitors of proteinase B, completely prevent the limited proteolysis of FBPase a by the vacuole lysate. Chymostatin and the endogenous proteinase inhibitor IB from yeast, both known as specific inhibitors of proteinase B, completely prevent the limited proteolysis of FBPase a by the vacuole lysate. Studies in the laboratory of Earl Stadtman have pointed to a possible biological significance of oxidative inactivation of glutamine synthetase from Escherichia coli by mixed function oxidation reactions. Interestingly, oxidative modification of one histidine residue per subunit of glutamine synthetase parallels the loss of its activity.