Detection of an enzyme bound γ‐glutamyl acyl ester of carbamyl phosphate synthetase of <i>Escherichia coli</i>

Lusty, Carol J.

doi:10.1016/0014-5793(92)80959-k

Cited by 32 publications

(25 citation statements)

References 17 publications

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“…All glutamineutilizing CPS II and Cps III contain Cys 301, His 385 and Glu 387 at the C-terminal GAT domain (Fig. 1), where the cysteine of the Cys-His-Glu triad carries out a nucleophilic attack on the amide carbonyl of glutamine to release ammonia (Chaparian and Evans 1991;Lusty 1992;Miles et al 1998;Thoden et al 1998 (Rubino et al 1986;Miran et al 1991;Huang and Raushel 1999), suggested that Cys and His functioned as a catalytic dyad for glutamine hydrolysis in the GAT site of Escherichia coli CPS (eCPS), whereas Glu of the GAT is not critical for eCPS activity. Non-glutamine-utilizing CPS I also share the two-domain GAT and four-domain SYN structure (Powers-Lee and Corina 1986; Marshall andFahien 1988, Rodriguez et al 1989) but they, with the exception of CPS I of R. catesbeiana, contain Ser 301, His 385 and Glu 387 at the C-terminal GAT domain (Fig.…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization and mRNA expression of carbamoyl phosphate synthetase III in the liver of the African lungfish, Protopterus annectens, during aestivation or exposure to ammonia

et al. 2011

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This study aimed to obtain the full sequence of carbamoyl phosphate synthetase III (cps III) from, and to determine the mRNA expression of cps III in, the liver of P. annectens during aestivation in air, hypoxia or mud, or exposure to environmental ammonia (100 mmol l(-1) NH(4)Cl). The complete coding cDNA sequence of cps III from the liver of P. annectens consisted of 4530 bp, which coded for 1,510 amino acids with an estimated molecular mass of 166.1 kDa. The Cps III of P. annectens consisted of a mitochondrial targeting sequence of 44 amino acid residues, a GAT domain spanning from tyrosine 45 to isoleucine 414, and a methylglyoxal synthase-like domain spanning from valine 433 to arginine 1513. Two cysteine residues (cysteine 1337 and cysteine 1347) that are characteristic of N-acetylglutamate dependency were also present. The critical Cys-His-Glu catalytic triad (cysteine 301, histidine 385 and glutamate 387) together with methionine 302 and glutamine 305 affirmed that P. annectens expressed Cps III and not Cps I. A comparison of the translated amino acid sequence of Cps III from P. annectens with CPS sequences from other animals revealed that it shared the highest similarity with elasmobranch Cps III. A phylogenetic analysis indicates that P. annectens CPS III could have evolved from Cps III of elasmobranchs. Indeed, Cps III from P. annectens used mainly glutamine as the substrate, and its activity decreased significantly when glutamine and ammonia were included together in the assay system. There were significant increases (9- to 12-fold) in the mRNA expression of cps III in the liver of fish during the induction phase (days 3 and 6) of aestivation in air. Aestivation in hypoxia or in mud had a delayed effect on the increase in the mRNA expression of cps III, which extended beyond the induction phase of aestivation, reiterating the importance of differentiating effects that are intrinsic to aestivation from those intrinsic to hypoxia. Furthermore, results from this study confirmed that environmental ammonia exposure led to a significant increase in the mRNA expression of cps III in the liver of P. annectens, alluding to the important functional role of urea not only as a product of ammonia detoxification but also as a putative internal cue for aestivation.

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Section: Discussionmentioning

confidence: 99%

Molecular characterization and mRNA expression of carbamoyl phosphate synthetase III in the liver of the African lungfish, Protopterus annectens, during aestivation or exposure to ammonia

et al. 2011

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“…glycinamidine synthetase and the carbamoyl-P synthetase adducts could be attacked by exogenous ammonia to regenerate glutamine [36,39] and by hydroxylamine to release pyrrolidone carboxylic acid as k-glutamyl hydroxamate and its cyclic derivative [36,72]; the failure of carbamoyl-P synthetase mutant Cys269Ser to form such an adduct was indicative of catalytic Cys 269 involvement. Consistent with the absence of acceptor substrate in these experiments, the kinetic parameters for the formation of this adduct have been found close to those of carbamoyl-P synthetase glutaminase activity, showing that its hydrolysis is the glutaminase rate-limiting step [39].…”

Section: M-shmentioning

confidence: 99%

The mechanism of glutamine-dependent amidotransferases

Massière¹,

Badet‐Denisot²

1998

CMLS, Cell. Mol. Life Sci.

201

213

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Glutamine-dependent amidotransferases have been known for more than 30 years. The mechanism by which these enzymes generate ammonia from the glutamine amide nitrogen and transfer it to seven different chemical classes of acceptors has been the subject of intense scrutiny for the last 5 years. The increasing number of biochemical and structural studies dealing with amidotransferases and with mechanistically related enzymes has disclosed the dichotomy of the mechanisms within these enzymes for achieving the glutamine amide bond cleavage. Some of them use a catalytic Cys/His/Glu triad similar to serine protease, whereas the aminoterminal cysteine of the others is believed to play the same function. The transfer of ammonia from the glutamine site to the acceptor site which must operate in a concerted manner has been demonstrated in two cases to involve channelling but is still matter of investigation.

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“…6) exhibits typical saturation kinetics, and when extrapolated to saturating ATP (Table II) showed a 12-fold increase in the GLNase activity. This coupling mechanism (26,54,55) ensures that the rate of glutamine hydrolysis matches the rate of production of carboxy phosphate.…”

Section: Construction and Expression Of The Recombinant Plasmids-mentioning

confidence: 99%

The Smallest Carbamoyl-phosphate Synthetase

Guy

Bouvier

Evans

1997

Journal of Biological Chemistry

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Escherichia coli carbamoyl-phosphate synthetase (CPSase) is comprised of a 40-kDa glutaminase (GLN) and a 120-kDa synthetase (CPS) subunit. The CPS subunit consists of two homologous domains, CPS.A and CPS.B, which catalyze the two different ATP-dependent partial reactions involved in carbamoyl phosphate synthesis. Sequence similarities and controlled proteolysis experiments suggest that the CPS subdomains consist, in turn, of three subdomains, designated A1, A2, A3 and B1, B2, B3 for CPS.A and CPS.B, respectively. Previous studies of individually cloned CPS.A and CPS.B from E. coli and mammalian CPSase have shown that homologous dimers of either of these "half-molecules" could catalyze all three reactions involved in ammoniadependent carbamoyl phosphate synthesis. Four smaller recombinant proteins were made for this study as follows: 1) A1-A2 in which the A3 subdomain was deleted from CPS.A, 2) B1-B2 lacking subdomain B3 of CPS.B, 3) the A2 subdomain of CPS.A, and 4) the B2 subdomain of CPS.B. When associated with the GLN subunit, A1-A2 and B1-B2 had both glutamine-and ammonia-dependent CPSase activities comparable to the wild-type protein. In contrast, the 27-kDa A2 and B2 recombinant proteins, which represent only 17% of the mass of the parent protein, were unable to use glutamine as a nitrogen donor, but the ammonia-dependent activity was enhanced 14 -16-fold. The hyperactivity suggests that A2 and B2 are the catalytic subdomains and that A1 and B1 are attenuation domains which suppress the intrinsically high activity and are required for the physical association with the GLN subunit.Carbamoyl-phosphate synthetase (CPSase, 1 EC 6.3.5.5) catalyzes the formation of carbamoyl phosphate from bicarbonate, NH 3 , usually derived from glutamine, and ATP (1, 2). The structure of CPSases can be quite diverse. For example, the monofunctional Escherichia coli CPSase (3, 4) consists of a 40-kDa glutaminase (GLN) subunit and a 120-kDa synthetase (CPS) subunit, whereas in its mammalian counterpart (5-7), the GLN and CPS domains are fused and are part of CAD, a multifunctional protein that also has aspartate transcarbamoylase and dihydroorotase activities. Despite the differences in structural organization, the amino acid sequences are clearly similar (8 -21) suggesting that all of these molecules are comprised of homologous domains and subdomains with analogous functions.The isolated GLN subunit of E. coli CPSase (3, 4) and the separately cloned GLN domain of CAD (22) hydrolyze glutamine (Reaction 1) and transfer ammonia to the CPS domain.All of the other partial reactions (Reactions 2-4) occur on the CPS domain or subunit (3, 4). The determination of the amino acid sequence of CPSase from many different organisms (8 -21) revealed that the CPS domain of these molecules invariably consists of two highly homologous halves, designated CPS.A and CPS.B. The two ATP-dependent partial reactions (Reactions 2 and 4) occur at different sites, and there is now convincing evidence (23-26) that CPS.A catalyzes the activation of bic...

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Detection of an enzyme bound γ‐glutamyl acyl ester of carbamyl phosphate synthetase of Escherichia coli

Cited by 32 publications

References 17 publications

Molecular characterization and mRNA expression of carbamoyl phosphate synthetase III in the liver of the African lungfish, Protopterus annectens, during aestivation or exposure to ammonia

Molecular characterization and mRNA expression of carbamoyl phosphate synthetase III in the liver of the African lungfish, Protopterus annectens, during aestivation or exposure to ammonia

The mechanism of glutamine-dependent amidotransferases

The Smallest Carbamoyl-phosphate Synthetase

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