Differential dynamic response of ATP-grasp domains within carbamoyl phosphate synthetase

CHED 818

Serena Hageman and Jason L. Johnson. Department of Chemistry and Physics, Southwestern Oklahoma State University, 100 Campus Drive, Weatherford, OK 73096
Carbamoyl phosphate synthetase is a heterodimer, with gene duplication giving rise to two ATP binding domains separated by over 40Å, having 40% sequence identity, and exhibiting strong structural overlap. During catalysis, the ATP-binding domains act in tandem. Within domain one, ATP reacts with bicarbonate to generate carboxyphosphate, signaling the hydrolysis of glutamine and release of ammonia from the small subunit. Attack by ammonia on carboxyphosphate generates carbamate, which tunnels to domain two and reacts with a second ATP to produce carbamoyl phosphate. Separate binding sites also exist for the allosteric activator ornithine and inhibitor UMP. How the conformations of the two ATP-grasp domains differentially respond to such synchronizing and regulatory signals has to this point been unknown. Here, we introduce tryptophans into parallel positions within the two active sites and report their unique steady-state and time-resolved fluorescence response to ligand binding. Supported by NIH-INBRE Grant #P20RR016478-04.