CRYSTAL-STRUCTURE OF THE COMPLEX OF UMP CMP KINASE FROM DICTYOSTELIUM-DISCOIDEUM AND THE BISUBSTRATE INHIBITOR P-1-(5'-ADENOSYL) P-5-(5'-URIDYL) PENTAPHOSPHATE (UP(5)A) AND MG2+ AT 2.2 ANGSTROM - IMPLICATIONS FOR WATER-MEDIATED SPECIFICITY/

The three-dimensional structure of the UMP/CMP kinase (UK) from the sl ime mold Dictyostelium discoideum complexed with the specific and asym metric bisubstrate inhibitor P-1-(5'-adenosyl) P-5-(5'-uridyl) pentaph osphate (UP(5)A) has been determined at a resolution of 2.2 Angstrom. The structure of the enzyme, which has up to 41% sequence homology wit h known adenylate kinases (AK), represents a closed conformation with the flexible monophosphate binding domain (NMP site) being closed over the uridyl moiety of the dinucleotide. Two water molecules were found within hydrogen-bonding distance to the uracil base. The key residue for the positioning and stabilization of those water molecules appears to be asparagine 97, a residue that is highly specific for AK-homolog ous UMP kinases, but is almost invariably a glutamine in adenylate kin ases. Other residues in this region are highly conserved among AK-rela ted NMP kinases. The catalytic Mg2+ ion is coordinated with octahedral geometry to four water molecules and two oxygens of the phosphate cha in of UP(5)A but has no direct interactions with the protein. The comp arison of the geometry of the UKdicty. UP(5)A . Mg(2+)complex with the previously reported structure of the UKyeast. ADP . ADP complex [Mull er-Dieckmann & Schulz (1994) J. Mol. Biol. 236. 361-367] suggests that UP(5)A in our structure mimics an ADP . Mg . UDP biproduct inhibitor rather than an ATP . Mg . UMP bisubstrate inhibitor.