Direct demonstration of carbamoyl phosphate formation on the C‐terminal domain of carbamoyl phosphate synthetase

Abstract

Carbamoyl phosphate synthetase synchronizes the utilization of two ATP molecules at duplicated ATPgrasp folds to catalyze carbamoyl phosphate formation. To define the dedicated functional role played by each of the two ATP sites, we have carried out pulse/labeling studies using the synthetases from Aquifex aeolicus and Methanococcus jannaschii, hyperthermophilic organisms that encode the two ATP-grasp folds on separate subunits. These studies allowed us to differentially label each active site with [␥-32 P]ATP and determine the fate of the labeled ␥-phosphate in the synthetase reaction. Our results provide the first direct demonstration that enzyme-catalyzed transfer of phosphate from ATP to carbamate occurs on the more C-terminal of the two ATP-grasp folds. These findings rule out one mechanism proposed for carbamoyl phosphate synthetase, where one ATP acts as a molecular switch, and provide additional support for a sequential reaction mechanism where the ␥-phosphate groups of both ATP molecules are transferred to reactants. CP synthesis by subunit C in our single turnover pulse/chase assays did not require subunit N, but subunit N was required for detectable CP synthesis in the traditional continuous assay. These findings suggest that cross-talk between domain N and C is required for product release from subunit C.Keywords: carbamoyl phosphate synthetase; ATP; ATP-grasp; arginine; glutamine; pyrimidine Synthesis of carbamoyl phosphate (CP) by carbamoyl phosphate synthetase (CPS) requires the coordinated utilization of two molecules of ATP per reaction cycle, as well as one molecule each of bicarbonate and ammonia (free or derived from glutamine through reaction on the glutamine amidotransferase domain of CPS) (Meister 1989). The ATP molecules react at two CPS domains (termed N for the one closest to the N terminus and C for the one closest to the C terminus) that share sequence identity and appear to have resulted from an ancestral gene duplication event (Lusty et al. 1983). The X-ray structure of Escherichia coli CPS (eCPS) demonstrated that both domains contain ATP-grasp folds and that the two folds are superimposable, with a root mean square deviation (RMSD) of 1.1 Å for 255 equivalent ␣ carbons (Thoden et al. 1997(Thoden et al. , 1999b. Despite the strong structural similarity of the two ATP-utilizing domains, a wide variety of studies have shown that each of the two ATP molecules plays a dedicated and unique role in the synchronized synthesis of ATP (Meister 1989;Javid-Majd et al. 2000).A multi-step CPS mechanism (Fig. 1) that was suggested by biochemical studies (Meister 1989) has been utilized to assign functions to the domains of the solved eCPS structure Reprint requests to: Susan Powers-Lee, Department of Biology, Northeastern University, Boston, MA 02115, USA; e-mail: spl@neu.edu; fax: (617) 373-3724.Abbreviations: aCPS, Aquifex aeolicus carbamoyl phosphate synthetase; cm, carbamate; CP, carbamoyl phosphate; CPS, carbamoyl phosphate synthetase; CPSs, plural form for carbamoyl phosphate synth...

Mechanisms of carbon fixation and engineering for increased carbon fixation in cyanobacteria

“…Carboxyphosphate reacts with ammonia (generated from glutamine) to form carbamate. The Cterminus of the small subunit catalyzes the reaction to form carbamoylphosphate using carbamate and ATP as substrates [66].…”

Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii

“…While the structure, function, and mechanisms of CPS catalysis and regulation have been well characterized for E. coli CPS (Thoden et al, 1997; Mora et al, 1999; Thoden et al, 1999b; Fresquet et al, 2000; Huang and Raushel, 2000a, 2000b, 2000c; Miles and Raushel, 2000), the larger CPSII polypeptides of eukaryotes are not yet well characterized at both the functional and structural level (Davidson et al, 1993; Graves et al, 2000; Fox and Bzik, 2003; Simmons et al, 2004; Kothe et al, 2005). Here, we report functional complementation of uracil auxotrophy in T. gondii based on CPSII minigenes.…”

Human carbamoyl‐phosphate synthetase: Insight into N‐acetylglutamate interaction and the functional effects of a common single nucleotide polymorphism

“…At the domain B active site, transfer of the ATP B g-phosphate group to bicarbonate yields ADP and the activated intermediate carboxyphosphate; ammonia (free or derived from glutamine) then reacts with carboxyphosphate to yield carbamate and P i . At the domain C active site, transfer of the ATP C g-phosphate group to carbamate yields ADP and CP (Kothe et al 2005;Thoden et al 1997). The 150-residue domain D ¶ and the 136-residue domain D contain interfaces for self-association (Thoden et al 1997(Thoden et al , 1999.…”