Characterization of the reactive sulfhydryl groups in carbamyl phosphate synthetase of Escherichia coli

Foley, Ronald; Poon, James; Anderson, Paul N.

doi:10.1021/bi00800a034

Cited by 33 publications

(35 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The two-ATP dependent partial reactions catalyzed by E. coli CPSase exhibit different catalytic and regulatory properties (4) and chemical modification (30,66) and inhibitor studies (67), suggest that they occur at distinct sites. Moreover, pulse-chase experiments (68 -70) with the E. coli CPSase and steady state kinetic studies of both E. coli (71) and mammalian (72) enzymes suggest that CPSase can bind 2 mol of ATP simultaneously.…”

Section: Discussionmentioning

confidence: 99%

Function of the Major Synthetase Subdomains of Carbamyl-phosphate Synthetase

Guy

Evans

1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

The amidotransferase domain (GLNase) of mammalian carbamyl-phosphate synthetase II hydrolyzes glutamine and transfers ammonia to the synthetase domain where carbamyl phosphate is formed in a three-step reaction sequence. The synthetase domain consists of two homologous subdomains, CPS.A and CPS.B. Recent studies suggest that CPS.A catalyzes the initial ATP dependent-activation of bicarbonate, whereas CPS.B uses a second ATP to form carbamyl phosphate. To establish the function of these substructural elements, we have cloned and expressed the mammalian protein and its subdomains in Escherichia coli. Recombinant CPSase (GLNase-CPS.A-CPS.B) was found to be fully functional. Two other proteins were made; the first consisted of only GLNase and CPS.A, whereas the second lacked CPS.A and had the GLNase domain fused directly to CPS.B. Remarkably, both proteins catalyzed the entire series of reactions involved in glutamine-dependent carbamyl phosphate synthesis. The stoichiometry, like that of the native enzyme, was 2 mol of ATP utilized per mol of carbamyl phosphate formed. GLN-CPS.B is allosterically regulated, whereas GLN-CPS.A was insensitive to effectors, a result consistent with evidence showing that allosteric effectors bind to CPS.B. These properties are not peculiar to the mammalian protein, because the separately cloned CPS.A subdomain of the E. coli enzyme was also found to catalyze carbamyl phosphate synthesis. Gel filtration chromatography and chemical cross-linking studies showed that these molecules are dimers, a structural organization that may be a prerequisite for the overall reaction. Thus, the homologous CPS.A and CPS.B subdomains are functionally equivalent, although in the native enzyme they may have different functions resulting from their juxtaposition relative to the other components in the complex.

show abstract

Section: Discussionmentioning

confidence: 99%

Function of the Major Synthetase Subdomains of Carbamyl-phosphate Synthetase

Guy

Evans

1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Enzyme preparations thought to be homogeneous (specific activity 280 pmoles of carbamyl phosphate formed/mg/hr at 37O (45) give a range of S values on velocity sedimentation because of self-association (46,47); equilibration between the several species, particularly in the presence of phosphate buffer, explains the variations in S values (48.49). In Tris or veronal buffers, pH 7.8, a single, stable species is obtained having an s20,w value of 7.3 S and a molecular weight, estimated by b. Molecular Weight and Subunit Composition.…”

Section: Carbamyl Phosphate Synthetase From E Colimentioning

confidence: 99%

Enzymes of Arginine and Urea Systhesis

Ratner¹

1973

Advances in Enzymology - And Related Areas of Molecular Biology

View full text Add to dashboard Cite

“…Escherichia coli CPS consists of a large subunit (CPS domains) with binding sites for substrates and allosteric effectors, and an associated small subunit glutamine amidotransferase (GATase) belonging to the Class I amidotransferase superfamily that catalyzes the hydrolysis of glutamine to glutamate and ammonia (Beckwith et al, 1962; Foley et al, 1971; Raushel et al, 1978; Thoden et al, 1999b). In contrast, the eukaryotic CPSII has an N-terminal GATase that is fused to the CPS polypeptide, as well as possessing fused C-terminal domains that catalyze subsequent steps in pyrimidine biosynthesis (Jones, 1980; Davis, 1986; Davidson et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

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

Fox

Ristuccia

Bzik

2009

International Journal for Parasitology

View full text Add to dashboard Cite

New treatments need to be developed for the significant human diseases of toxoplasmosis and malaria to circumvent problems with current treatments and drug resistance. Apicomplexan parasites causing these lethal diseases are deficient in pyrimidine salvage suggesting that selective inhibition of de novo pyrimidine biosynthesis can lead to a severe loss of UMP and dTMP pools thereby inhibiting parasite RNA and DNA synthesis. Disruption of Toxoplasma gondii carbamoyl phosphate synthetase II (CPSII) induces a severe uracil auxotrophy with no detectable parasite replication in vitro and complete attenuation of virulence in mice. Here we show that a CPSII cDNA minigene efficiently complements the uracil auxotrophy of CPSII deficient mutants restoring parasite growth and virulence. Our complementation assays reveal that engineered mutations within or proximal to the catalytic triad of the N-terminal glutamine amidotransferase (GATase) domain inactivate the complementation activity of T. gondii CPSII and demonstrate a critical dependence on the apicomplexan CPSII GATase domain in vivo. Surprisingly, indels present within the T. gondii CPSII GATase domain as well as the C-terminal allosteric regulatory domain are found to be essential. In addition several mutations directed at residues implicated in allosteric regulation in Escherichia coli CPS either abolish or markedly suppress complementation and further define the functional importance of the allosteric regulatory region. Collectively, these findings identify novel features of T. gondii CPSII as potential parasite-selective targets for drug development.

show abstract

Characterization of the reactive sulfhydryl groups in carbamyl phosphate synthetase of Escherichia coli

Cited by 33 publications

References 13 publications

Function of the Major Synthetase Subdomains of Carbamyl-phosphate Synthetase

Function of the Major Synthetase Subdomains of Carbamyl-phosphate Synthetase

Enzymes of Arginine and Urea Systhesis

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

Contact Info

Product

Resources

About