Access to the carbamate tunnel of carbamoyl phosphate synthetase

Kim, Jungwook; Raushel, Frank M.

doi:10.1016/j.abb.2004.02.031

Cited by 16 publications

(11 citation statements)

References 19 publications

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“…The existence of the first tunnel, allowing sequestered movement of uncharged ammonia from the amidotransferase domain to domain N, has been well documented in CPS and other amidotransferases (Raushel et al 2003). Documented functioning of the second tunnel, proposed to allow sequestered movement of carbamate from domain N to domain C, has remained elusive (Kim and Raushel 2004), but the operation of the sequential reaction mechanism supported by the present data would be consistent with such an interior closed channel.…”

Section: Hyperthermophilic Cpss Provide a Critical Mechanistic Probesupporting

confidence: 53%

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

et al. 2005

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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...

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Section: Hyperthermophilic Cpss Provide a Critical Mechanistic Probesupporting

confidence: 53%

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

et al. 2005

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“…The similarity must extend to the existence in CPSI of the tunnel that in E. coli CPS extends over nearly 100 Å , pervading the enzyme practically from end to end. 22 Indeed, the tunnel wall residues are highly conserved among CPSs, 37,39,52 including CPSI. Further, human and E. coli CPSs catalyse carbamoyl phosphate synthesis from ammonia in the same three steps involving two unstable water-sensitive intermediates, and thus the tunnel is essential, at least for the intraenzymatic migration of carbamate (the carbamate tunnel) between the two phosphorylation sites.…”

Section: Final Remarksmentioning

confidence: 99%

Understanding Carbamoyl Phosphate Synthetase Deficiency: Impact of Clinical Mutations on Enzyme Functionality

Yefimenko

Fresquet

Marco-Marı́n

et al. 2005

Journal of Molecular Biology

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“…Cys-269 and His-353 of the glutaminase subunit are two residues essential for the hydrolysis of glutamine that passes through a thioester intermediate (Miran et al 1991; Khedouri et al 1966; Pinkus and Meister 1972; Anderson and Carlson 1975; Rubino et al 1986; Mullins et al 1991; Thoden et al 1998, 1999a; Huang and Raushel 1999; Rishavy et al 2000). From the structure, it appears that the three active sites in the enzyme are located far apart but connected by an intramolecular tunnel of 96 Å that leads from the glutamine binding site on the small subunit over the carboxyphosphate site (about 45 Å away) all the way to the carbamate phosphorylation site (another ~ 40 Å long tunnel) (Thoden et al 1997; Huang and Raushel 2000a, b; Huang et al 2001; Kim et al 2002; Kim and Raushel 2004a, b; Fan et al 2008, 2009; Lund et al 2010). It is proposed that tunneling of the highly unstable reaction intermediates (Fig.…”

Section: E Coli Cpsase: Reaction Intermediates and Enzyme Structurementioning

confidence: 99%

Regulation of carbamoylphosphate synthesis in Escherichia coli: an amazing metabolite at the crossroad of arginine and pyrimidine biosynthesis

Charlier

Minh

Roovers³

2018

Amino Acids

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In all organisms, carbamoylphosphate (CP) is a precursor common to the synthesis of arginine and pyrimidines. In Escherichia coli and most other Gram-negative bacteria, CP is produced by a single enzyme, carbamoylphosphate synthase (CPSase), encoded by the carAB operon. This particular situation poses a question of basic physiological interest: what are the metabolic controls coordinating the synthesis and distribution of this high-energy substance in view of the needs of both pathways? The study of the mechanisms has revealed unexpected moonlighting gene regulatory activities of enzymes and functional links between mechanisms as diverse as gene regulation and site-specific DNA recombination. At the level of enzyme production, various regulatory mechanisms were found to cooperate in a particularly intricate transcriptional control of a pair of tandem promoters. Transcription initiation is modulated by an interplay of several allosteric DNA-binding transcription factors using effector molecules from three different pathways (arginine, pyrimidines, purines), nucleoid-associated factors (NAPs), trigger enzymes (enzymes with a second unlinked gene regulatory function), DNA remodeling (bending and wrapping), UTP-dependent reiterative transcription initiation, and stringent control by the alarmone ppGpp. At the enzyme level, CPSase activity is tightly controlled by allosteric effectors originating from different pathways: an inhibitor (UMP) and two activators (ornithine and IMP) that antagonize the inhibitory effect of UMP. Furthermore, it is worth noticing that all reaction intermediates in the production of CP are extremely reactive and unstable, and protected by tunneling through a 96 Å long internal channel.

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Access to the carbamate tunnel of carbamoyl phosphate synthetase

Cited by 16 publications

References 19 publications

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

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

Understanding Carbamoyl Phosphate Synthetase Deficiency: Impact of Clinical Mutations on Enzyme Functionality

Regulation of carbamoylphosphate synthesis in Escherichia coli: an amazing metabolite at the crossroad of arginine and pyrimidine biosynthesis

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