GLYCEROL KINASE FROM ESCHERICHIA-COLI AND AN ALA65-]THR MUTANT - THE CRYSTAL-STRUCTURES REVEAL CONFORMATIONAL-CHANGES WITH IMPLICATIONS FORALLOSTERIC REGULATION

Background: Glycerol kinase (GK) from Escherichia coli is a velocity-m odulated (V system) enzyme that has three allosteric effecters with in dependent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarr ier protein IIA(Glc); and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetram ers, GK is a member of a superfamily of ATPases that share a common AT Pase domain and are thought to undergo a large conformational change a s an intrinsic step in their catalytic cycle, Members of this family i nclude actin, hexokinase and the heat shock protein hsc70. Results: We report here the crystal structures of GK and a mutant of GK (Ala65--> Thr) in complex with glycerol and ADP, Crystals of both enzymes contai n the same 222 symmetric tetramer. The functional dimer is identical t o that described previously for the IIA(Glc)-GK complex structure, The tetramer interface is significantly different, however, with a relati ve 22.3 degrees rotation and 6.34 Angstrom translation of one function al dimer. The overall monomer structure is unchanged except for two re gions: the IIA(Glc)-binding site undergoes a structural rearrangement and residues 230-236 become ordered and bind orthophosphate at the tet ramer interface. We also report the structure of a second mutant of GK (Ile474-->Asp) in complex with IIA(Glc); this complex crystallized is omorphously to the wild type IIA(Glc)-GK complex. Site-directed mutant s of GK with substitutions at the IIA(Glc)-binding site show significa ntly altered kinetic and regulatory properties, suggesting that the co nformation of the binding site is linked to the regulation of activity . Conclusions: We conclude that the new tetramer structure presented h ere is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consisten t with it being part of the FBP-binding site, Mutational analysis and the structure of the IIA(Glc)-GK(Ile474-->Asp) complex suggest the con formational transition of the IIA(Glc)-binding site to be an essential aspect of IIA(Glc) regulation.