A GLIMPSE AT AN ENZYME REACTION INTERMEDIATE: THE ATP-Mg²⁺-OXALATE TERNARY COMPLEX OF ESCHERICHIA COLI PHOSPHOENOLPYRUVATE CARBOXYKINASE. Leslie W. Tari, Allan Matte, Umarani Pugazhenthi, Hughes Goldie and Louis T. J. Delbaere. Departments of Biochemistry and Microbiology, University of Saskatchewan, Saskatoon Saskatchewan, Canada S7N 0W0

Phosphoenolpyruvate carboxykinase (PCK; E. C. 4.1.1.49) is a key metabolic enzyme which catalyzes the reaction representing the first committed step in the diversion of tricarboxylic acid cycle intermediates towards gluconeogenesis, the conversion of oxaloacetate to phosphoenolpyruvate. By molecular replacement, the crystal structure of adenosine triphosphate (ATP)-magnesium-oxalate bound PCK from Escherichia coli (M_r 59583, 540 residues) has been solved, using the structure of native PCK as a search model. Refinement by energy-restrained least-squares, simulated annealing, manual model building and map re-fitting converged at an R-factor of 19.5% using 35508 6.0-1.8 Å data (76% complete). ATP binding induces a large hinge-like rotation of the N- and C-terminal domains which closes the active site cleft. PCK possesses a novel nucleotide binding fold, particularly in the adenine-binding region, where the formation of a cis backbone torsion angle in a loop glycine promotes intimate contacts between the adenine-binding loop and adenine, while stabilizing a syn-conformation of the purine base. This complex represents a reaction intermediate analog along the pathway of the conversion of oxaloacetate to phosphoenolpyruvate, and provides insight into the mechanistic details of the chemical reaction catalyzed by this enzyme.