Synchronization of the Three Reaction Centers within Carbamoyl Phosphate Synthetase

Abstract

Carbamoyl phosphate synthetase from E. coli catalyzes the synthesis of carbamoyl phosphate through a series of four reactions occurring at three active sites connected by a molecular tunnel of 100 A. To understand the mechanism for coordination and synchronization among the active sites, the pre-steady-state time courses for the formation of phosphate, ADP, glutamate, and carbamoyl phosphate were determined. When bicarbonate and ATP were rapidly mixed with CPS, a stoichiometric burst of acid-labile phosphate and ADP was observed with a formation rate constant of 1100 min(-)(1). The burst phase was followed by a linear steady-state phase with a rate constant of 12 min(-)(1). When glutamine or ammonia was added to the initial reaction mixture, the magnitude and the rate of formation of the burst phase for either phosphate or ADP were unchanged, but the rate constant for the linear steady-state phase increased to an average value of 78 min(-)(1). These results demonstrate that the initial phosphorylation of bicarbonate is independent of the binding or hydrolysis of glutamine. The pre-steady-state time course for the hydrolysis of glutamine in the absence of ATP exhibited a burst of glutamate formation with a rate constant of 4 min(-)(1) when the reaction was quenched with base. In the presence of ATP and bicarbonate, the rate constant for the formation of the burst of glutamate was 1100 min(-)(1). The hydrolysis of ATP thus enhanced the hydrolysis of glutamine by a factor of 275, but there was no effect by glutamine on the initial phosphorylation of bicarbonate. The pre-steady-state time course for the formation of carbamoyl phosphate was linear with an overall rate constant of 72 min(-)(1). The absence of an initial burst of carbamoyl phosphate formation eliminates product release as a rate-determining step for CPS. Overall, these results have been interpreted to be consistent with a mechanism whereby the phosphorylation of bicarbonate serves as the initial trigger for the rest of the reaction cascade. The formation of the carboxy phosphate intermediate within the large subunit must induce a conformational change to the active site of the small subunit that enhances the hydrolysis of glutamine. Thus, ammonia is not released into the molecular tunnel until the activated bicarbonate is ready to form carbamate. The rate-limiting step for the steady-state assembly of carbamoyl phosphate is either the formation, migration, or phosphorylation of the carbamate intermediate.

Detection of Protein-Protein Interactions in the Alkanesulfonate Monooxygenase System from Escherichia coli

“…One classic example in support of a substrate channeling mechanism is found in tryptophan synthase from Salmonella enterica serovar Typhimurium, where the metabolic intermediate (indole) is thought to channel from the active site of the ␣ subunit to the active site of the ␤ subunit (1, 10, 20, 22). Substrate channeling has also been well characterized in carbamoyl phosphate synthase, a protein with three distinct active sites (17,21,26). It has been fully established that reduced flavin is a very reactive intermediate and is easily oxidized in the presence of molecular oxygen, generating species toxic to the cells.…”

Subunit Interactions and Glutamine Utilization by Escherichia coli Imidazole Glycerol Phosphate Synthase

“…It is logical that these events are temporally controlled so that when the acceptor site is occupied with an activated substrate, the enzyme specificity is enhanced (21). The modular evolutionary process proposed for the triad GATs assumes distinct origins for the glutamine and acceptor domains (33); consequently, the molecular details for the ammonia transfer event are likely different for every GAT.…”

Gain of Glutaminase Function in Mutants of the Ammonia-specific Frog Carbamoyl Phosphate Synthetase

“…To determine ammonium-dependent ADP formation, 30 mM NH 4 Cl was included in the reaction mixture; to determine glutamine-dependent ADP formation, 10 mM glutamine was included. Although only the unprotonated form of ammonia is a substrate for CPSs (18,19), the data are presented as the total of [NH 4 ϩ ] ϩ [NH 3 ] because it is the level of NH 4 Cl that is varied during the experiments. Under the conditions of the present studies, NH 3 represents about 4% of the total NH 4 ϩ added to the solution (18).…”

“…Formation of the high energy intermediate CP requires concomitant cleavage of two molecules of ATP to form two ADPs (19,21). In ammonia-dependent ADP formation assays, K258L and K258L/E261Q displayed wild type kinetic parameters for ammonia and ATP usage ( Table I).…”