W0093

Structural Studies on Carbamoyl Phosphate Synthetase from Escherichia Coli. James B. Thoden, Frank M. Raushel, Ivan Rayment, and Hazel. M. Holden, Institute for Enzyme Research and Department of Biochemistry, University of Wisconsin, Madison, WI 53705 and Department of Chemistry, Texas A&M University, College Station, Texas 77843

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, MgATP, and glutamine or ammonia. The enzyme, as isolated from E. coli, is an [alpha],[beta]-heterodimer consisting of a large subunit (Mr 118000) that catalyzes two phosphorylations, and a small subunit (Mr 42000) that hydrolyzes glutamine. In its active form, CPS is an [alpha]₄[beta]₄-tetramer. Formation of carbamoyl phosphate requires at least four separate chemical reactions: (1) phosphorylation of bicarbonate with MgATP to generate carboxyphosphate; (2) hydrolysis of glutamine to glutamate and ammonia; (3) formation of carbamate through nucleophilic attack of the ammonia on the carboxyphosphate; and (4) phosphorylation of carbamate by a second molecule of MgATP to form carbamoyl phosphate. Amazingly, the three active sites are separated by a total distance of approximately 100 Å, requiring that the reactive and unstable intermediates formed at one active site must be channeled through the interior of the protein for reaction at the next active site without being released into the bulk solvent.

CPS was crystallized in the presence of MnCl₂, KCl, ADP, and ornithine at pH 7.4 with PEG-8000 and tetraethylammonium chloride as precipitants. The crystal belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 153.1 Å, b = 163.9 Å, and c = 332.8 Å and one tetramer per asymmetric unit. The structure was solved "in-house" using phases obtained from four heavy atom derivatives in combination with solvent flattening and molecular averaging to 2.8 Å resolution. The resolution has since been extended up to 1.8 Å for various substrate complexes by collecting x-ray data at both the SSRL and APS synchrotron facilities. The structures of these complexes will be described.