[3] Glutamine-dependent carbamyl-phosphate synthetase (Escherichia coli); preparation of subunits

Trotta, Paul P.; Burt, Michael; Pinkus, Lawrence M.; Estis, Leonard F.; Haschemeyer, Rudy H.; Meister, Alton

doi:10.1016/s0076-6879(78)51005-7

Cited by 18 publications

(21 citation statements)

References 18 publications

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“…Using the densitometric estimates of CPS content in the corresponding extracts, the estimated speci¢c activity of the pure enzyme is 4.76 Wmol/min/mg for wild-type CPS and 1.45, 1.20 and 1.46 Wmol/min/mg for the Ser-948-Ala, Lys-954-Ala and Thr-974-Ala mutant forms, respectively. The speci¢c activity estimated for the wild-type enzyme is within the expected range for pure E. coli CPS [18,19]. The lower estimated speci¢c activity of the mutant forms of CPS does not re£ect substantial di¡erences in enzyme solubility, since the mutant forms, similarly to wildtype CPS, appeared mainly in the supernatant after centrifugation of the sonicates of the transformed cells.…”

Section: Resultssupporting

confidence: 55%

Localization of the site for the nucleotide effectors ofEscherichia colicarbamoyl phosphate synthetase using site‐directed mutagenesis

et al. 1999

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Replacement by alanine of Ser-948, Thr-974 and Lys-954 of Escherichia coli carbamoyl phosphate synthetase (CPS) shows that these residues are involved in binding the allosteric inhibitor UMP and the activator IMP. The mutant CPSs are active in vivo and in vitro and exhibit normal activation by ornithine, but the modulation by both UMP and IMP is either lost or diminished. The results demonstrate that the sites for UMP and IMP overlap and that the activator ornithine binds elsewhere. Since the mutated residues were found in the crystal structure of CPS near a bound phosphate, Ser-948, Thr-974 and Lys-954 bind the phosphate moiety of UMP and IMP.z 1999 Federation of European Biochemical Societies.

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Section: Resultssupporting

confidence: 55%

Localization of the site for the nucleotide effectors ofEscherichia colicarbamoyl phosphate synthetase using site‐directed mutagenesis

et al. 1999

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“…Both the ammonia-dependent, and glutamine-dependent activities of the reconstituted CPSase regained the usual sensitivity to the feedback inhibitor, suggesting that the light subunit functions in allosteric control by UMP. No equivalent data has been published for E. coli and the conclusion that the heavy subunit contains the feedback inhibition site [33] is based on inhibition by high UMP concentrations of the reverse partial reaction: MgADP + carbamoyl phosphate 4 MgATP + carbamate. The observed inhibition could be the result of competition with carbamoyl phosphate in a similar manner to the inhibition by CTP and other phosphorylated compounds of the catalytic subunit of aspartate carbamoyltransferase [43].…”

Section: Discussionmentioning

confidence: 99%

Nucleotide sequence of the carA gene and regulation of the carAB operon in Salmonella typhimurium

Kilstrup¹,

Lu²,

Abdelal³

et al. 1988

European Journal of Biochemistry

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The carAB operon of Salmonella typhimurium encoding carbamoyl-phosphate synthetase (CPSase) has been cloned, and the nucleotide sequence of the first gene of the operon, carA, together with 760 base pairs of the 5'-flanking region was determined. The product of the carA gene is the small subunit of CPSase. It catalyzes the transfer of the amide group from glutamine to an NH,-site on the heavy subunit. Primer extension and S1 nuclease mapping of in vivo carAB transcripts revealed that transcription is similar to that of Escherichia co/i [Piette, J. et al. (1984) Proc. Nut1 Acad. Sci. U S A 81, 4134-41381 in its initiation at two promoters, PI and P2, controlled by pyrimidines and arginine, respectively. The arginine control is mediated through binding to the arginine repressor (argR). The involvement of titratable regulatory elements is indicated by the escape from both arginine and pyrimidine control, when the operon is present in multicopies on a plasmid. Measurements of CPSase levels in mutants which allows independent manipulation of the intracellular uracil and cytosine nucleotide pools show, that both uracil and cytosine nucleotides are required for full repression and that limitation of either nucleotide results in derepression of CPSase synthesis. Deletion analyses indicate that regions upstream of the PI promoter are required for normal expression from this promoter but not from P2.Carbamoyl-phosphate synthetase (CPSase) encoded by the carAB (pyrA) operon in Salmonella typhimurium [3] and Escherichia coli [4], catalyzes the synthesis of carbamoyl-phosphate from ATP, bicarbonate and glutamine. It plays a dual role in metabolism by supplying carbamoyl phosphate for both the pyrimidine and the arginine biosynthetic pathways. In accordance with its physiological function, CPSase is allosterically regulated by intermediates of both pathways. The pyrimidine nucleotide UMP inhibits CPSase activity while ornithine, an intermediate in the arginine biosynthetic pathway stimulates it [5].The enzyme from S. typhimurium consists of two nonidentical subunits. The light subunit (Mr = 45000), encoded by the first gene in the operon (carA), converts the amide nitrogen of glutamine to ammonia. The heavy subunit ( M , = IlOOOO), encoded by the second gene of the operon (carB), catalyzes the formation of carbamoyl-phosphate from ammonia, carbon dioxide and ATP, it also contains the binding sites for ornithine. The ammonia-dependent activity of the isolated heavy subunit is only slightly inhibited by high concentrations of UMP and the reconstituted holoenzyme regains sensitivity to this feedback inhibitor [3]. [I]. ,In S. typhimuriurn the homologous locus is designated pyrA [2]. In order to be consistent we propose that the E. coli nomenclature is adopted for the genes encoding S. typhimurium carbamoyl-phosphate synthetase.Expression of carAB is subject to cumulative repression by pyrimidines and arginine. In E. coli [4, 6, 71 transcription of the operon was shown to be initiated at tandem promoters. Transcription from th...

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“…Carbamoylphosphate synthetase from P. aeruginosa is similar in many respects to the enzymes from Escherichia coli [3,4] and Salmonella typhimurium [1,2]. The three enzymes have identical subunit composition and are subject to feed-back inhibition by UMP and activation by ornithine.…”

Section: Discussionmentioning

confidence: 99%

“…The finding that carbamoylphosphate synthetase from P. aeruginosa does not associate in the presence of positive allosteric effectors represents a significant difference from carbamoylphosphate synthetase of enteric bacteria [2,4,5,28]. Meister and coworkers have proposed that the self-association, promoted by ornithine or MgATP, is a mechanism for allosteric activation of the enzyme and that enzyme concentration may play a major role in regulation of activity in vivo [28].…”

Section: Discussionmentioning

confidence: 99%

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Carbamoylphosphate Synthetase from Pseudomonas aeruginosa

Abdelal¹,

Bussey²,

Vickers³

1983

European Journal of Biochemistry

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Carbamoylphosphate synthetase from Pseudomonas ueruginosa is subject to repression by pyrimidines and significant derepression by limitation of arginine or pyrimidines. Carbamoylphosphate synthetase was purified to homogeneity from a derepressed strain of P. aeruginosa. The molecular weight of the enzyme was estimated to be 168 000 by sucrose gradient ultracentrifugation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme is composed of two non-identical subunits with molecular weights of 122000 and 44000. Cross-linking the enzyme prior to electrophoresis yielded an additional band corresponding to a molecular weight of 165000, showing that the enzyme is composed of one of each subunit. The enzyme utilized either glutamine ( K , 0.15 mM) or NH3 (K, 17 mM) and requires free Mg2+ for maximal activity with the optimal level between 4 mM and 10 mM. Hill plots of MgATP saturation data yielded coefficients of 1.2 and 1.4 at pH 8.0 and 8.5, respectively. A Hill equation was derived on the assumptions that MgATP binds at the same time to two distinct sub-sites as was shown to be the case for carbamoylphosphate synthetase from Escherichia coli and that these sub-sites are strictly non-interacting. The resulting theoretical Hill coefficients correspond very closely to the experimental coefficients. Carbamoylphosphate synthetase activity was subject to activation by ornithine and N-acetylornithine and feedback inhibition by UMP. Carbamoylphosphate synthetase from P. aerugino.ra does not associate under all conditions examined, establishing that self-association does not play a role in regulation of enzyme activity as suggested by other workers for the enzyme from E. coli.Carbamoylphosphate is an intermediate in the biosynthesis of arginine and pyrimidines. In enteric bacteria [l -51 it is synthesized by a single enzyme, carbamoylphosphate synthetase, which catalyzes the following reaction :Ammonia can replace glutamine as a nitrogen donor in the reaction but the affinity for ammonia is much less than that for glutamine; the latter substrate is considered the physiological nitrogen donor. The enteric enzymes [l -51 are all subject to cumulative repression by arginine and a pyrimidine compound and to feedback control: U M P inhibits activity and ornithine stimulates it. The combined effects provide an efficient mechanism for controlling the supply of carbamoylphosphate for arginine and pyrimidine biosynthesis.Examination of carbamoylphosphate metabolism in Pseudomonas aeruginosa is of particular interest in view of the significant differences that distinguish arginine metabolism in this organism from that in enteric bacteria [6,7]. These differences include the ability of P. ueruginosu and other fluorescent pseudomonads to degrade arginine via the arginine deiminase pathway (Fig. 1). This pathway involves the activity of carbamate kinase which catalyzes the phosphorylation of ADP by carbamoylphosphate [7] according to the following reaction :The possible presence of two enzyme...

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[3] Glutamine-dependent carbamyl-phosphate synthetase (Escherichia coli); preparation of subunits

Cited by 18 publications

References 18 publications

Localization of the site for the nucleotide effectors ofEscherichia colicarbamoyl phosphate synthetase using site‐directed mutagenesis

Localization of the site for the nucleotide effectors ofEscherichia colicarbamoyl phosphate synthetase using site‐directed mutagenesis

Nucleotide sequence of the carA gene and regulation of the carAB operon in Salmonella typhimurium

Carbamoylphosphate Synthetase from Pseudomonas aeruginosa

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