A switch in the kinase domain of rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatas e plays an essential role in the regulation of glucose metabolism by both p roducing and degrading Fru-2,6-P-2 via its distinct catalytic activities. T he 6-PF-2-K and Fru-2,6-P(2)ase active sites are located in separate domain s of the enzyme. The kinase domain is structurally related to the superfami ly of mononucleotide binding proteins that includes adenylate kinase and th e G-proteins, We have determined three new structures of the enzymatic mono mer, each with a different ligand in the ATP binding site of the 6-PF-2-K d omain (AMP-PNP, PO4, and water). A comparison of these three new structures with the ATP gamma S-bound 6-PF-2-K domain reveals a rearrangement of a he lix that is dependent on the ligand bound in the ATP binding site of the en zyme. This helix motion dramatically alters the position of a catalytic res idue (Lys172), This catalytic cation is analogous to the Arg residue donate d by the rasGAP protein, and the Arg residue at the core of the GTP or GDP sensing switch motion seen in the heterotrimeric G-proteins, In addition, a succinate molecule is observed in the Fru-6-P binding site. Kinetic analys is of succinate inhibition of the 6-PF-2-K reaction is consistent with the structural findings, and suggests a mechanism for feedback inhibition of gl ycolysis by citric acid cycle intermediates. Alterations in the 6-PF-2-K ki netics of several proteins mutated near both the switch and the succinate b inding site suggest a mode of communication between the ATP- and F6P bindin g sites. Together with these kinetic data, these new structures provide ins ights into the mechanism of the 6-PF-2-K activity of this important bifunct ional enzyme.