Molecular cloning, sequence analysis, and expression of a human liver cDNA coding for fructose-6-P,2-kinase:fructose-2,6-bisphosphatase.

A cDNA coding for 378 amino acids from the C-terminus of the human liver bifunctional enzyme, Fructose-6-phosphate,2-kinase:Fructose-2,6-bisphosphatase was isolated, sequenced, and expressed in E. coli K38. The expressed protein, identified by specific immunoassay, showed Fru 2,6-bisphosphatase activity but no Fru 6-P,2-kinase activity, demonstrating directly that the Fru 2,6-bisphosphatase activity resides in the C-terminal region. The Km for Fru 2,6-P2 was 4.3 microM. Fru 6-P was a noncompetitive inhibitor (Ki = 2.9 microM), and formed a phosphorylated intermediate when incubated with Fru 2,6[2-32P]P2. The subunit Mr of the enzyme was 36,600, and the active enzyme showed Mr = 37,000 by gel filtration.