REGULATION OF RAT 6-PHOSPHOFRUCTO-2-KINASE FRUCTOSE-2,6-BISPHOSPHATASE - ROLE OF THE NH2-TERMINAL REGION

The role of the NH2-terminal region of the liver and skeletal muscle 6 -phosphofructo-2-kinase/fructose 2,6-bisphosphatases was investigated, as well that of a mutant of the liver isoform lacking the first 22 am ino acids, by the overexpression of these enzymes in Escherichia coli and the comparison of their kinetic properties. The muscle isoform and the deletion mutant had K(m) values for fructose 6-phosphate which we re 50- and 20-fold higher, respectively, than that of the liver isofor m, and the bisphosphatase maximal velocity of the liver deletion mutan t was 4-fold higher than that of the native liver isoform. Phosphoryla tion of the liver isoform increased bisphosphatase activity by 2-3-fol d and the K(m) for fructose 6-phosphate of the 6-phosphofructo-2-kinas e by 10-15-fold, but these kinetic effects were greatly diminished for the deletion mutant despite equivalent phosphorylation by cAMP-depend ent protein kinase. Arg-173 of the skeletal muscle isoform was found t o be functionally equivalent to the residue corresponding to the essen tial fructose 6- phosphate binding residue of the liver kinase domain, Arg-195. The results suggest that 1) the NH2-terminal regions of the liver and skeletal muscle isoforms are important determinants of fruct ose 6-phosphate affinity, and 2) the initial 22 amino acids of the liv er isoform exert an inhibitory influence on the bisphosphatase and med iate, at least in part, the response of both activities of the enzyme to cAMP-dependent phosphorylation.