Function of the Major Synthetase Subdomains of Carbamyl-phosphate Synthetase

Guy, Hedeel I.; Evans, David R.

doi:10.1074/jbc.271.23.13762

Cited by 36 publications

(45 citation statements)

References 71 publications

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“…Once a particular subunit would have synthesized carboxyphosphate, the second one would become committed to the synthesis of CP. This is in striking agreement with the functional model proposed by Guy and Evans (27) on the basis of the observation that homodimers of either the CPS.A or CPS.B subdomains from E. coli and mammalian CPSs, when coupled to the glutaminase subunit, can carry out the complete set of reactions for CP synthesis. Our hypothesis thus assumes that the first step in the evolution of CPS from CK consisted in mutations of a primeval CK gene giving a product similar to the enzyme described here.…”

Section: Discussionsupporting

confidence: 77%

The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus , a missing link in the evolution of carbamoyl phosphate biosynthesis

Durbecq

Legrain

Roovers

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Microbial carbamoyl phosphate synthetases (CPS) use glutamine as nitrogen donor and are composed of two subunits (or domains), one exhibiting glutaminase activity, the other able to synthesize carbamoyl phosphate (CP) from bicarbonate, ATP, and ammonia. The pseudodimeric organization of this synthetase suggested that it has evolved by duplication of a smaller kinase, possibly a carbamate kinase (CK). In contrast to other prokaryotes the hyperthermophilic archaeon Pyrococcus furiosus was found to synthesize CP by using ammonia and not glutamine. We have purified the cognate enzyme and found it to be a dimer of two identical subunits of M r 32,000. Its thermostability is considerable, 50% activity being retained after 1 h at 100°C or 3 h at 95°C. The corresponding gene was cloned by PCR and found to present about 50% amino acid identity with known CKs. The stoichiometry of the reaction (two ATP consumed per CP synthesized) and the ability of the enzyme to catalyze at high rate a bicarbonate-dependent ATPase reaction however clearly distinguish P. furiosus CPS from ordinary CKs. Thus the CPS of P. furiosus could represent a primeval step in the evolution of CPS from CK. Our results suggest that the first event in this evolution was the emergence of a primeval synthetase composed of subunits able to synthesize both carboxyphosphate and CP; this step would have preceded the duplication assumed to have generated the two subdomains of modern CPSs. The gene coding for this CK-like CPS was called cpkA.Carbamoyl phosphate (CP) is a key metabolite in nitrogen metabolism because it is a precursor of both arginine and the pyrimidines. Three classes of carbamoyl phosphate synthetases (CPS, EC 6.3.5.5) have been recognized on the basis of their substrate specificity-whether glutamine or ammonia is used as the nitrogen donor-and their requirement for N-acetyl-Lglutamate as allosteric effector (reviewed in refs. 1 and 2). Despite this apparent diversity, all three forms of CPS consist of two entities, a glutaminase domain (or subunit) and a synthetase domain (or subunit) that catalyzes ATP-dependent synthesis of CP from ammonia and bicarbonate. In prokaryotic and fungal arginine-specific CPSs the two functions are fulfilled by separate polypeptides, whereas in other organisms the homologous parts of the molecule are domains within the same polypeptide. The synthesis of CP by the synthetase domain or subunit of CPS proceeds in three consecutive steps (1, 3, 4); two ATP molecules are consumed in the process (Fig. 1).Analysis of the DNA sequence encoding the synthetase subunit of Escherichia coli (the carB gene) revealed considerable similarity between the N-and C-terminal halves of the protein, and it was proposed that the synthetase gene had evolved by duplication of a smaller ancestral gene-possibly coding for a carbamate kinase (CK) (5). CKs (EC 2.7.2.2) are homodimers composed of subunits of 31-37 kDa (6). As carbamate is in chemical equilibrium with bicarbonate and ammonia, CK can synthesize CP from HCO 3 Ϫ , NH 3 and...

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Section: Discussionsupporting

confidence: 77%

The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus , a missing link in the evolution of carbamoyl phosphate biosynthesis

Durbecq

Legrain

Roovers

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…If each homologous half catalyzes a phosphorylation step, the two halves This mechanism was based on the observation that CPS catashould be very close to allow the transfer of intermediates, but lyzes partial reactions of bicarbonate-dependent ATP cleavage the X-ray structure of the E. coli enzyme has demonstrated a and of ATP synthesis from ADP and carbamoyl phosphate large space between the two halves [19]. Furthermore, the isowhich were interpreted to reflect, respectively, the step of bicarlated expression of only a half [20] or even of its 27-kDa catabonate phosphorylation (reaction 2) and the reversal of the step lytic core [21] allows homodimerization of the protein and synof carbamate phosphorylation (reaction 4) [1]. However, there thesis of carbamoyl phosphate, excluding the need for structural are doubts about the significance of the partial reaction of ATP specialization that was implicit in the assignement of a phossynthesis, which, given the similarities of carbamoyl phosphate phorylation step to each half of the large subunit.…”

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confidence: 99%

Mechanism of carbamoyl phosphate synthetase from Escherichia coli

Rubio¹,

Llorente²,

Britton³

1998

European Journal of Biochemistry

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The conflicting data on the binding of the two molecules of ATP that are involved in the overall reaction catalyzed by carbamoyl-phosphate synthetase (CPS) of Escherichia coli, and a mechanism recently proposed for this reaction, has led us to reexamine ATP binding using pulse/chase techniques. With [γ-32 P]ATP and bicarbonate in the pulse solution, there is a positive intercept at zero time of approximately 1 mol P i /mol CPS in the plot of 32 P i formation against time, irrespective of whether the incubation is terminated by the addition of acid or by addition of a chase solution containing glutamine, excess unlabeled ATP and bicarbonate. The intercept is decreased to about 50% if the excess unlabeled ATP is added prior to the addition of the glutamine. These are the expected results if the intercept reflects the reversible formation of enzyme-bound ADP and carboxyphosphate. Approximately 0.6 mol carbamoyl [ 32 P]phosphate/mol enzyme is formed in these experiments when the pulse step is terminated by addition to the chase solution. The ATP molecule that provides the phosphoryl group of carbamoyl phosphate, therefore, also binds to the enzyme in the absence of ammonia or glutamine and reacts in the chase to give carbamoyl phosphate before it can dissociate from the enzyme. At 1 mM ATP, the binding of both ATP molecules is essentially complete at 2.5 s, but the dissociation of the ATP that yields carbamoyl phosphate is extremely slow (t 1/2 of about 6 min at 22°C; HCO Ϫ 3 , 40 mM), although it is faster in the absence of bicarbonate. The extreme sequestration from the aqueous environment of this ATP allows the enzyme-ATP complex to be separated from the surrounding ATP by centrifugal gel filtration. After two successive steps of gel filtration through Sephadex G-50 equilibrated with unlabeled ATP and bicarbonate, the majority of the radioactivity remaining in the solution is bound to the enzyme and is released as [γ-32 P]ATP if acid is added, or is converted to carbamoyl [ 32 P]phosphate by addition to chase solution, without concomitant release of 32 P i . K ϩ is necessary in the pulse solution, but not in the chase solution, to demonstrate this binding. These findings and other confirmatory experiments demonstrate conclusively that, in the presence of K ϩ , both ATP molecules bind to the enzyme in the absence of ammonia or glutamine. The bound ATP that yields P i in the overall reaction is replaced relatively rapidly by exchange and by hydrolysis in the bicarbonate-dependent ATPase activity of the enzyme, whereas the bound ATP that provides the phosphoryl group of carbamoyl phosphate is replaced very slowly. The temporal pattern of carbamoyl [ 32 P]phosphate formation from [γ-32 P]ATP, in pulse/chase experiments in which a small concentration of ammonia is added to the pulse solution, shows that, in the normal enzyme reaction, this last ATP molecule binds to the enzyme before ammonia.These findings exclude a recently proposed mechanism [Kothe, M., Eroglu, B., Mazza, H., Samudera, H. & Powers- Lee, S. (1997) Pr...

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“…The resulting ATP saturation curve was hyperbolic, although significant substrate inhibition was apparent at ATP concentrations exceeding 2 mM. The ATP saturation curve of CPS.B exhibits similar inhibition at high ATP concentrations (41). The K m for ATP for the CAD autophosphorylation reaction was 0.33 Ϯ 0.026 mM.…”

Section: Resultsmentioning

confidence: 82%

Autophosphorylation of the Mammalian Multifunctional Protein That Initiates de Novo Pyrimidine Biosynthesis

Sigoillot

Evans

Guy

2002

Journal of Biological Chemistry

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CAD, a large multifunctional protein that carries carbamoyl phosphate synthetase (CPSase), aspartate transcarbamoylase, and dihydroorotase activities, catalyzes the first three steps of de novo pyrimidine biosynthesis in mammalian cells. The CPSase component, which catalyzes the initial, rate-limiting step, exhibits complex regulatory mechanisms involving allosteric effectors and phosphorylation that control the flux of metabolites through the pathway. Incubation of CAD with ATP in the absence of exogenous kinases resulted in the incorporation of 1 mol of P i /mol of CAD monomer. Mass spectrometry analysis of tryptic digests showed that Thr 1037 located within the CAD CPS.B subdomain was specifically modified. The reaction is specific for MgATP, ADP was a competitive inhibitor, and the native tertiary structure of the protein was required. Phosphorylation occurred after denaturation, further purification of CAD by SDS gel electrophoresis, and renaturation on a nitrocellulose membrane, strongly suggesting that phosphate incorporation resulted from an intrinsic kinase activity and was not the result of contaminating kinases. Chemical modification with the ATP analog, 5-pfluorosulfonylbenzoyladenosine, showed that one or both of the active sites that catalyze the ATP-dependent partial reactions are also involved in autophosphorylation. The rate of phosphorylation was dependent on the concentration of CAD, indicating that the reaction was, at least in part, intermolecular. Autophosphorylation resulted in a 2-fold increase in CPSase activity, an increased sensitivity to the feedback inhibitor UTP, and decreased allosteric activation by 5-phosphoribosyl-1-pyrophosphate, functional changes that were distinctly different from those resulting from phosphorylation by either the protein kinase A or mitogen-activated protein kinase cascades.CAD (1-3) is a multifunctional protein that catalyzes the first three steps of the de novo pyrimidine biosynthetic pathway in mammalian cells. The protein consists of multiple copies of a 243-kDa polypeptide organized into discrete domains (Fig. 1A) that have glutamine-dependent carbamoyl phosphate synthetase (CPSase), 1 aspartate transcarbamoylase (ATCase) and dihydroorotase (DHOase) activities.The CPSase activity, the first committed and rate-limiting step in the pathway, is the locus of control. Most CPSases share a common catalytic mechanism (4) in which the synthesis of carbamoyl phosphate proceeds through a complex series of partial reactions catalyzed by different functional domains.CPSases also have homologous domain structures. Glutamine hydrolysis (Reaction 1) occurs on the 40-kDa glutaminase domain or subunit. The synthetase (CPS) domain or subunit consists of two homologous 60-kDa subdomains, CPS.A and CPS.B. The activation of bicarbonate and the subsequent reaction with NH 3 (Reactions 2 and 3) occurs on CPS.A, whereas the phosphorylation of carbamate to form carbamoyl phosphate (Reaction 4) occurs on CPS.B (5). Lusty recognized that the CPS domain (6) evolved by gene duplic...

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Function of the Major Synthetase Subdomains of Carbamyl-phosphate Synthetase

Cited by 36 publications

References 71 publications

The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus , a missing link in the evolution of carbamoyl phosphate biosynthesis

The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus , a missing link in the evolution of carbamoyl phosphate biosynthesis

Mechanism of carbamoyl phosphate synthetase from Escherichia coli

Autophosphorylation of the Mammalian Multifunctional Protein That Initiates de Novo Pyrimidine Biosynthesis

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