Mutations in the charged residues of the amino terminus of rat liver fructose 6-phosphate,2-kinase: Fructose 2,6-bisphosphatase: Effects on regulation

Amino and carboxyl termini of the bifunctional enzyme Fru 6-P,2-kinase:Fru 2,6-bisphosphatase regulate the relative activities of the kinase/phosphata se. The N-terminus of the rat liver bifunctional enzyme is highly basic, co ntaining a protein kinase A phosphorylation site that regulates these enzym e activities in a reciprocal manner. To determine the role of charged resid ues in the N-terminal peptide, mutant enzymes were constructed in which the se residues were altered to residues carrying opposite charges, and the eff ect on the catalytic properties, thermal lability, and susceptibility to tr ypsin digestion and phosphorylation by protein kinase A was determined. Mos t of these mutations decreased K-cat/K-ATP and/or k(cat)/KFru 6-P of the ki nase and increased k(cat)/K-Fru 2,K-6-P2 of the phosphatase. These mutant e nzymes were more susceptible to trypsin digestion, phosphorylation by prote in kinase A, and thermal inactivation. In general, the effect was greater w ith amino acid residues located more distant from the N-terminus. The resul ting changes were not as large as observed with the phosphorylated enzyme. Mutation of Ser22 to Pro produced large changes in the kinetic properties c omparable to those of phosphorylation, suggesting that the flexible region of the N-terminus containing five serines (Ser20 to S24) is essential for t he enzyme activities. These results indicated that the charged residues as well as Ser20-Ser24 in the N-terminus of the liver Fru 6-P,2-kinase:Fru 2,6 -Pase are essential in the allosteric regulation and probably involved in i nteractions with the catalytic domains that induce a conformation that has high Fru 6-P,2-kinase and low Fru 2,6-Pase activities. Any disruption of th is N-terminal interaction results in inhibition of the kinase and activatio n of the phosphatase. (C) 1999 Academic Press.