Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.3.5.5 (CPS)
1,262 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The relationship between intramitochondrial and extramitochondrial ATP-utilizing systems and the intramitochondrial ATP/ADP ratio was studied in isolated rat-liver mitochondria. Citrulline synthesis was used as an intramitochondrial ATP-utilizing system, and glucose-6-phosphate synthesis as an extramitochondrial ATP-utilizing system. The intramitochondrial ATP/ADP ratio was manipulated in three ways: with succinate and different concentrations of malonate and/or hexokinase; with 2-oxoglutarate (plus oligomycin) and different concentrations of hexokinase; and with added ATP in uncoupled mitochondria (oligomycin present). 2. Under all conditions used, citrulline synthesis was strictly correlated with the bulk intramitochondrial ATP/ADP ratio. 3. The curve relating citrulline synthesis and intramitochondrial ATP/ADP was shifted towards lower ATP/ADP ratios when the activity of carbamoyl-phosphate synthetase was enhanced by increasing the mitochondrial content of N-acetylglutamate. 4. It is concluded that under the experimental conditions used the intramitochondrial adenine nucleotides behave as a homogeneous pool.
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PMID:Relationship between the rate of citrulline synthesis and bulk changes in the intramitochondrial ATP/ADP ratio in rat-liver mitochondria. 720 12

Purified carbamoyl-phosphate synthetase of rat liver is shown to exist in a state of rapid, reversible monomer-dimer equilibrium. The allosteric activator N-acetyl-L-glutamate displaces the equilibrium toward monomer formation. This effect is observed over a range of initial protein concentrations of 0.02-5 mg/mL. Measurements of Stokes radii by analytical gel chromatography indicate that at concentrations less than 0.1 mg/mL at 25 degrees C in the presence of all the substrates the enzyme exists as a monomer of 160000 molecular weight. A gel chromatographic method was developed to identify the active form of carbamoyl-phosphate synthetase. On the basis of analysis of the ADP boundary formed during gel chromatography, the monomer is established to be catalytically active. Active enzyme centrifugation studies confirm that the monomer is a reactive species and suggest that the dimer also functions catalytically. Under the conditions of the usual enzyme assay, carbamoyl-phosphate synthetase is mainly in the monomer form. Activation by acetylglutamate can occur at the level of the monomer and is not coupled to dissociation since the enzyme dissociates at low concentrations even in the absence of acetylglutamate. The stoichiometry of the association is observed directly in the electron microscope. The dimensions of the negatively stained particles of the enzyme in the presence or absence of substrates correspond to monomers and dimers, assuming the molecule to be a prolate ellipse. The number of monomers observed in the presence of substrate represents 86% of the total number of enzyme molecules. The average molecular weight calculated from the numbers of particles seen in negatively stained specimens of carbamoyl-phosphate synthetase is 182000. Electron microscope studies provide independent evidence for monomer--dimer interactions and show that under the conditions examined the enzyme is mainly in the monomer form.
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PMID:Catalytically active monomer and dimer forms of rat liver carbamoyl-phosphate synthetase. 727 72

Three conserved histidine residues, His-243, His-781, and His-788, located within the large subunit of carbamoyl phosphate synthetase from Escherichia coli were identified by sequence identity comparisons. These three histidine residues were individually mutated to asparagine residues. The H243N mutant enzyme was found to be critical for carbamoyl phosphate synthesis as the mutant protein was unable to synthesize carbamoyl phosphate at a significant rate (< 1/1500). By analysis of the effects of this mutation on the partial reactions catalyzed by CPS, it was determined that this mutation blocked the formation of the carbamate intermediate from carboxyphosphate and ammonia. The H781N mutant enzyme had an order of magnitude reduction for both the rate of carbamoyl phosphate formation and ATP synthesis which is consistent with the proposal that the carboxyl-terminal half of the large subunit is primarily involved in the phosphorylation of the putative carbamate intermediate. This mutation also reduced the effects of the allosteric activator ornithine on the Km parameters for ATP in the overall biosynthetic reaction and ADP in the ATP synthesis reaction. The H788N mutant enzyme is a functional protein which maintains the ability to synthesize carbamoyl phosphate at a rate comparable to that of the wild-type enzyme. The effects of this mutation are 10-fold reductions of the ATP synthetase and the bicarbonate-dependent ATPase activities with substantial increases in the Km values for ATP in the full biosynthetic reaction and for ADP in the ATP synthesis reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential roles for three conserved histidine residues within the large subunit of carbamoyl phosphate synthetase. 841 43

Carbamate kinase (CK) catalyzes the reversible reaction NH2COO- + ATP <--> NHCOOPO3(2-) + ADP, serving to synthesize ATP from carbamoyl phosphate in those microorganisms that derive energy from anaerobic arginine degradation via the arginine dihydrolase pathway. We report here the cloning and sequencing of the CK gene from Enterococcus faecalis and Enterococcus faecium and we demonstrate that the amino acid sequence of CK is identical in the two species. The enzyme, expressed and isolated from Escherichia coli using simple purification procedures, was used to generate crystals suitable for X-ray studies and to investigate the utilization by CK of bicarbonate and other carbamate analogs. CK had a bicarbonate-dependent ATPase activity and, therefore, is able to synthesize carboxyphosphate, an unstable compound that is an intermediate in the reactions catalyzed by carbamoyl-phosphate synthetase (CPS) and by biotin carboxylase. Other functional similarities with CPS include the utilization of acetate by CK with a similarly high Km and the similar Km values of CK for carbamate and of CPS for bicarbonate. Enterococcal CK was inhibited by adenosine(5')pentaphospho(5')adenosine (Ap5A) and Ap6A and, less powerfully, by Ap4A, whereas Ap3A is essentially non-inhibitory. Thus, inhibition by Ap5A seems not to be a valid criterion to differentiate between CK and CPS, for the two enzymes can be inhibited by Ap5A. All these results support the relatedness of CK and CPS. Finally, we used limited proteolysis: (a) to localize the epitopes for monoclonal antibodies obtained against CK; (b) to demonstrate the importance of the C-terminus for enzyme activity; and (c) to show that Arg158 is highly exposed and may be essential for activity. Comparison of the sequence of CK with known protein sequences demonstrates considerable similarity of CK with bacterial N-acetylglutamate kinases, strongly suggesting that these two enzymes may share a similar structure and the same catalytic mechanism.
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PMID:Carbamate kinase from Enterococcus faecalis and Enterococcus faecium--cloning of the genes, studies on the enzyme expressed in Escherichia coli, and sequence similarity with N-acetyl-L-glutamate kinase. 957 87

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 [gamma-32P]ATP and bicarbonate in the pulse solution, there is a positive intercept at zero time of approximately 1 mol Pi/mol CPS in the plot of 32Pi 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 [32P]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 degrees C; HCO3-, 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 [gamma-32P]ATP if acid is added, or is converted to carbamoyl [32P]phosphate by addition to chase solution, without concomitant release of 32Pi. K+ 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+, both ATP molecules bind to the enzyme in the absence of ammonia or glutamine. The bound ATP that yields Pi 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 [32P]phosphate formation from [gamma-32P]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) Proc. Natl Acad. Sci. USA 94, 12348-12353] in which a single molecule of ATP bound at the catalytic center phosphorylates bicarbonate and provides the phosphoryl group of carbamoyl phosphate. A mechanism in which a single ATP molecule binds, followed by the binding of bicarbonate and ammonia (from glutamine) and the release of Pi before the second molecule of ATP is bound is also excluded. We have previously reported very similar findings for carbamoyl-phosphate synthetase (ammonia), strongly suggesting that the different types of CPS share a common mechanism. The virtual sequestration of the ATP that provides the phosphoryl group of carbamoyl phosphate is consistent with a palmate-binding site, with the nucleotide bound within a beta-sheet sandwich, and a loop closure mechanism triggered by the binding of bicarbonate or the formation of carboxyphosphate.
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PMID:Mechanism of carbamoyl phosphate synthetase from Escherichia coli--binding of the ATP molecules used in the reaction and sequestration by the enzyme of the ATP molecule that yields carbamoyl phosphate. 969 27

Carbamoyl phosphate synthetase catalyzes the formation of carbamoyl phosphate from one molecule of bicarbonate, two molecules of Mg2+ATP and one molecule of glutamine or ammonia depending upon the particular form of the enzyme under investigation. As isolated from Escherichia coli, the enzyme is an alpha,beta-heterodimer consisting of a small subunit that hydrolyzes glutamine and a large subunit that catalyzes the two required phosphorylation events. Here the three-dimensional structure of carbamoyl phosphate synthetase from E. coli refined to 2.1 A resolution with an R factor of 17.9% is described. The small subunit is distinctly bilobal with a catalytic triad (Cys269, His353 and Glu355) situated between the two structural domains. As observed in those enzymes belonging to the alpha/beta-hydrolase family, the active-site nucleophile, Cys269, is perched at the top of a tight turn. The large subunit consists of four structural units: the carboxyphosphate synthetic component, the oligomerization domain, the carbamoyl phosphate synthetic component and the allosteric domain. Both the carboxyphosphate and carbamoyl phosphate synthetic components bind Mn2+ADP. In the carboxyphosphate synthetic component, the two observed Mn2+ ions are both octahedrally coordinated by oxygen-containing ligands and are bridged by the carboxylate side chain of Glu299. Glu215 plays a key allosteric role by coordinating to the physiologically important potassium ion and hydrogen bonding to the ribose hydroxyl groups of ADP. In the carbamoyl phosphate synthetic component, the single observed Mn2+ ion is also octahedrally coordinated by oxygen-containing ligands and Glu761 plays a similar role to that of Glu215. The carboxyphosphate and carbamoyl phosphate synthetic components, while topologically equivalent, are structurally different, as would be expected in light of their separate biochemical functions.
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PMID:The structure of carbamoyl phosphate synthetase determined to 2.1 A resolution. 1008 90

Carbamoyl phosphate synthetase from E. coli catalyzes the synthesis of carbamoyl phosphate through a series of four reactions occurring at three active sites connected by a molecular tunnel of 100 A. To understand the mechanism for coordination and synchronization among the active sites, the pre-steady-state time courses for the formation of phosphate, ADP, glutamate, and carbamoyl phosphate were determined. When bicarbonate and ATP were rapidly mixed with CPS, a stoichiometric burst of acid-labile phosphate and ADP was observed with a formation rate constant of 1100 min(-)(1). The burst phase was followed by a linear steady-state phase with a rate constant of 12 min(-)(1). When glutamine or ammonia was added to the initial reaction mixture, the magnitude and the rate of formation of the burst phase for either phosphate or ADP were unchanged, but the rate constant for the linear steady-state phase increased to an average value of 78 min(-)(1). These results demonstrate that the initial phosphorylation of bicarbonate is independent of the binding or hydrolysis of glutamine. The pre-steady-state time course for the hydrolysis of glutamine in the absence of ATP exhibited a burst of glutamate formation with a rate constant of 4 min(-)(1) when the reaction was quenched with base. In the presence of ATP and bicarbonate, the rate constant for the formation of the burst of glutamate was 1100 min(-)(1). The hydrolysis of ATP thus enhanced the hydrolysis of glutamine by a factor of 275, but there was no effect by glutamine on the initial phosphorylation of bicarbonate. The pre-steady-state time course for the formation of carbamoyl phosphate was linear with an overall rate constant of 72 min(-)(1). The absence of an initial burst of carbamoyl phosphate formation eliminates product release as a rate-determining step for CPS. Overall, these results have been interpreted to be consistent with a mechanism whereby the phosphorylation of bicarbonate serves as the initial trigger for the rest of the reaction cascade. The formation of the carboxy phosphate intermediate within the large subunit must induce a conformational change to the active site of the small subunit that enhances the hydrolysis of glutamine. Thus, ammonia is not released into the molecular tunnel until the activated bicarbonate is ready to form carbamate. The rate-limiting step for the steady-state assembly of carbamoyl phosphate is either the formation, migration, or phosphorylation of the carbamate intermediate.
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PMID:Synchronization of the three reaction centers within carbamoyl phosphate synthetase. 1081 70

PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, functions in purine biosynthesis by catalyzing the formylation of glycinamide ribonucleotide through a catalytic mechanism requiring Mg(2+)ATP and formate. From previous x-ray diffraction analyses, it has been demonstrated that PurT transformylase from Escherichia coli belongs to the ATP-grasp superfamily of enzymes, which are characterized by three structural motifs referred to as the A-, B-, and C-domains. In all of the ATP-grasp enzymes studied to date, the adenosine nucleotide ligands are invariably wedged between the B- and C-domains, and in some cases, such as biotin carboxylase and carbamoyl phosphate synthetase, the B-domains move significantly upon nucleotide binding. Here we present a systematic and high-resolution structural investigation of PurT transformylase complexed with various adenosine nucleotides or nucleotide analogs including Mg(2+)ATP, Mg(2+)-5'-adenylylimidodiphosphate, Mg(2+)-beta,gamma-methyleneadenosine 5'-triphosphate, Mg(2+)ATPgammaS, or Mg(2+)ADP. Taken together, these studies indicate that the conformation of the so-called "T-loop," delineated by Lys-155 to Gln-165, is highly sensitive to the chemical identity of the nucleotide situated in the binding pocket. This sensitivity to nucleotide identity is in sharp contrast to that observed for the "P-loop"-containing enzymes, in which the conformation of the binding motif is virtually unchanged in the presence or absence of nucleotides.
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PMID:PurT-encoded glycinamide ribonucleotide transformylase. Accommodation of adenosine nucleotide analogs within the active site. 1195 35

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'-p-fluorosulfonylbenzoyladenosine, 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.
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PMID:Autophosphorylation of the mammalian multifunctional protein that initiates de novo pyrimidine biosynthesis. 1198 31

A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with inorganic phosphates, carbamoyl phosphate, the antiviral phosphonate foscarnet as well as formate is reported. The cytosolic isozyme hCA I was weakly inhibited by neutral phosphate, strongly inhibited by carbamoyl phosphate (K(I) of 9.4 microM), and activated by hydrogen- and dihydrogenphosphate, foscarnet and formate (best activator foscarnet, K(A)=12 microM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with carbamoyl phosphate showing a K(I) of 0.31 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by phosphates/phosphonates, showing a K(I) of 84 nM for PO(4)(3-), of 9.8 microM for HPO(4)(2-), and of 9.9 microM for carbamoyl phosphate. Foscarnet was the best inhibitor of this isozyme (K(I) of 0.82 mM) highly abundant in the kidneys, which may explain some of the renal side effects of the drug. The mitochondrial isozyme hCA V was weakly inhibited by all phosphates/phosphonates, except carbamoyl phosphate, which showed a K(I) of 8.5 microM. Thus, CA V cannot be the isozyme involved in the carbamoyl phosphate synthetase I biosynthetic reaction, as hypothesized earlier. Furthermore, the relative resistance of CA V to inhibition by inorganic phosphates suggests an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of such anions in these energy-converting organelles, where high amounts of ATP are produced by ATP synthetase, from ADP and inorganic phosphates. The transmembrane, tumor-associated isozyme hCA IX was on the other hand slightly inhibited by all these anions.
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PMID:Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with phosphates, carbamoyl phosphate, and the phosphonate antiviral drug foscarnet. 1550 Oct 37


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