AN EVOLUTIONARY FUSION OF TWO ENZYMES: STRUCTURE OF THE UNIQUE BIFUNCTIONAL ENZYME 6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE. Charles A. Hasemann^*, E. Istvan^#, K. Uyeda^#, and Johann Deisenhofer^#t, Depts. of Internal Medicine, Biochemistry^#, and the Howard Hughes Medical Inst, UT Southwestern Medical Center, Dallas, Texas 75235-8884

The evolutionary fusion of two protein domains with antagonistic catalytic activities has led to the unique bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6-PF-2-K/Fru-2,6-P₂ase). This enzyme indirectly controls the rate of glycolysis via the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P₂), a potent activator of phosphofructokinase (PFK). 6-PF-2-K/Fru2,6P₂ase is found as a homodimer of ~56 kD subunits in the cytosol of all eukaryotes. The need for glycolytic activity varies both with time and from tissue to tissue, and thus the activity of 6-PF-2-K/Fru2,6P₂ase regulated both in time, and in a tissue specific manner. Consequently, tissue specific isoforms of 6-PF-2-K/Fru2,6P₂ase have been identified (liver, muscle, heart, testis, and brain), each with a characteristic relative ratio of kinase to phosphatase activity, and each regulated by inhibitors (i.e., PEP, citrate, phosphoglycerate) which vary in concentration with the metabolic needs of the cell. The activities of the liver and heart COOH-terminal regulatory domains. The phosphorylation of the liver isozyme has been shown to coordinately modulate both the 6-PF-2-K and 6-PF-2-K/Fru2,6P₂ase activities, are fused in a single polypeptide.

We have recently solved by X-ray diffraction the 2.0Å resolution structure of the rat testis isozyme of 6-PF-2-K/Fru2,6P₂ase. As predicted by sequence homology, the Fru2,6P₂ase domain resembles the structure of yeast phosphoglycerate mutase, and these enzymes no doubt share a common catalytic mechanism involving a phosphohistidine intermediate. Surprisingly, the 6-PF-2-K domain does not resemble PFK as had been predicted, but instead has clearly evolved from the adenylate kinase family. This homology, coupled with the apparent lack of a nucleophile to activate the 2-hydroxyl of F6P, points to a mechanism of catalysis distinct from that of PFK. The domain interface in the monomer, and the orientation of the domains in the functional dimer, provide clues to the regulation by phosphorylation observed in the liver and heart isozymes.