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:4.1.1.6 (CAD)
4,420 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have measured the 'core' mammalian carbamoyl-phosphate synthetase II (CPSII) activity, using NH4Cl as the nitrogen-donating substrate and trapping carbamoyl phosphate as urea through its reaction with ammonium ions. When ATP and magnesium ion concentrations are close to those found in the cell, the substrate saturation curves for ammonia and bicarbonate are hyperbolic, giving Km (NH3) values of 166 microM at high ATP concentrations and 26 microM at low ATP concentrations, while the Km (bicarbonate) is 1.4 mM at both ATP concentrations used. These values for the Km (NH3) are lower than previously reported for CPS II, and closer to the values for the mitochondrial counterpart. The Km for ammonia and bicarbonate are not altered by phosphorylation of the multienzyme polypeptide CAD, which contains the first three enzyme activities of pyrimidine biosynthesis. The CPS II activity is lower with an excess of either ATP or magnesium ions, causing the apparently sigmoid dependence of activity upon ATP concentration to be enhanced at low concentrations of free magnesium ions. The feedback inhibitor, UTP, acts by stabilising a state with a low affinity for magnesium ions and for ATP. In the presence of the activator, 5-phosphoribosyl diphosphate (PRibPP), the enzyme has a higher affinity for magnesium ions and thus the ATP dependence of the activity is hyperbolic. Phosphorylation of CAD similarly activates the CPS II enzyme by increasing the affinity for magnesium ions and by pushing the equilibrium away from the low-affinity UTP-stabilised state. Using our improved assay procedure, we observe a very large activation by PRibPP of carbamoylphosphate synthesis at low concentrations of magnesium ions, and we find that unlike UTP, the activator PRibPP is able to act on the phosphorylated enzyme.
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PMID:Regulation of the mammalian carbamoyl-phosphate synthetase II by effectors and phosphorylation. Altered affinity for ATP and magnesium ions measured using the ammonia-dependent part reaction. 149 69

The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA) was used to chemically modify the ATP binding sites of the carbamyl phosphate synthetase domain of CAD, the multifunctional protein that catalyzes the first steps in mammalian pyrimidine biosynthesis. Reaction of CAD with FSBA resulted in the inactivation of the ammonia- and glutamine-dependent CPSase activities but had no effect on its glutaminase, aspartate transcarbamylase, or dihydroorotase activities. ATP protected CAD against inactivation by FSBA whereas the presence of the allosteric effectors UTP and PRPP afforded little protection, which suggests that the ATP binding sites were specifically labeled. The inactivation exhibited saturation behavior with respect to FSBA with a K1 of 0.93 mM. Of the two ATP-dependent partial activities of carbamyl phosphate synthetase, bicarbonate-dependent ATPase was inactivated more rapidly than the carbamyl phosphate dependent ATP synthetase, which indicates that these partial reactions occur at distinct ATP binding sites. The stoichiometry of [14C]FSBA labeling showed that only 0.4-0.5 mol of FSBA/mol of protein was required for complete inactivation. Incorporation of radiolabeled FSBA into CAD and subsequent proteolysis, gel electrophoresis, and fluorography demonstrated that only the carbamyl phosphate synthetase domain of CAD is labeled. Amino acid sequencing of the principal peaks resulting from tryptic digests of FSBA-modified CAD located the sites of FSBA modification in regions that exhibit high homology to ATP binding sites of other known proteins. Thus CAD has two ATP binding sites, one in each of the two highly homologous halves of the carbamyl phosphate domain which catalyze distinct ATP-dependent partial reactions in carbamyl phosphate synthesis.
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PMID:Identification of the ATP binding sites of the carbamyl phosphate synthetase domain of the Syrian hamster multifunctional protein CAD by affinity labeling with 5'-[p-(fluorosulfonyl)benzoyl]adenosine. 168

The trifunctional protein CAD, which contains the first three enzyme activities of pyrimidine nucleotide biosynthesis (carbamyl phosphate synthetase II, aspartate transcarbamylase and dihydro-orotase), is phosphorylated stoichiometrically by cyclic AMP-dependent protein kinase. Phosphorylation activates the ammonia-dependent carbamyl phosphate synthetase activity of the complex by reducing the apparent Km for ATP. This effect is particularly marked in the presence of the allosteric feedback inhibitor, UTP, when the apparent Km is reduced by greater than 4-fold. Inhibition by physiological concentrations of UTP is substantially relieved by phosphorylation. Cyclic AMP-dependent protein kinase phosphorylates two serine residues on the protein termed sites 1 and 2, and the primary structures of tryptic peptides containing these sites have been determined: Site 1: Arg-Leu-Ser(P)-Ser-Phe-Val-Thr-Lys Site 2: Ile-His-Arg-Ala-Ser(P)-Asp-Pro-Gly-Leu-Pro-Ala-Glu-Glu-Pro-Lys During the phosphorylation reaction, activation of the carbamyl phosphate synthetase shows a better correlation with occupancy of site 1 rather than site 2. Both phosphorylation and activation can be reversed using purified preparations of the catalytic subunits of protein phosphatases 1- and -2A, and inactivation also correlates better with dephosphorylation of site 1 rather than site 2. We believe this to be the first report that a key enzyme in nucleotide biosynthesis is regulated in a significant manner by reversible covalent modification. The physiological role of this phosphorylation in the stimulation of cell proliferation by growth factors and other mitogens is discussed.
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PMID:Phosphorylation and activation of hamster carbamyl phosphate synthetase II by cAMP-dependent protein kinase. A novel mechanism for regulation of pyrimidine nucleotide biosynthesis. 409 95

Carbamyl-phosphate synthetases from different organisms have similar catalytic mechanisms and amino acid sequences, but their structural organization, sub-unit structure, and mode of regulation can be very different. Escherichia coli carbamyl-phosphate synthetase (CPSase), a monofunctional protein consisting of amido-transferase and synthetase subunits, is allosterically inhibited by UMP and activated by NH3, IMP, and ornithine. In contrast, mammalian CPSase II, part of the large multifunctional polypeptide, CAD, is inhibited by UTP and activated by 5-phosphoribosyl-1-pyrophosphate (PRPP). Previous photoaffinity labeling studies of E. coli CPSase showed that allosteric effectors bind near the carboxyl-terminal end of the synthetase subunit. This region of the molecule may be a regulatory subdomain common to all CPSases. An E. coli mammalian hybrid CPSase gene has been constructed and expressed in E. coli. The hybrid consists of the E. coli CPSase synthetase catalytic subdomains, residues 1-900 of the 1073 residue polypeptide, fused to the amino-terminal end of the putative 190-residue regulatory subdomain of the mammalian protein. The hybrid CPSase had normal activity, but was no longer regulated by the prokaryotic allosteric effectors. Instead, the glutamine- and ammonia-dependent CPSase activities and both ATP-dependent partial reactions were activated by PRPP and inhibited by UTP, indicating that the binding sites of both of these ligands are located in a regulatory region at the carboxyl-terminal end of the CPSase domain of CAD. The apparent ligand dissociation constants and extent of inhibition by UTP are similar in the hybrid and the wild type mammalian protein, but PRPP binds 4-fold more weakly to the hybrid. The allosteric ligands affected the steady state kinetic parameters of the hybrid differently, suggesting that while the linkage between the catalytic and regulatory subdomains has been preserved, there may be qualitative differences in interdomain signal transmission. Nevertheless, switching prokaryotic and eukaryotic allosteric controls argues for remarkable conservation of structure and regulatory mechanisms in this family of proteins.
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PMID:Identification of the regulatory domain of the mammalian multifunctional protein CAD by the construction of an Escherichia coli hamster hybrid carbamyl-phosphate synthetase. 752 61

Our purpose was to assess the efficacy and safety of intravenous ATP for the acute termination of paroxysmal supraventricular tachycardia. There were 14 women and 10 men, aged 38 +/- 15 years. Three patients had evidence of structural heart disease (Ebstein's anomaly associated to atrial septal defect, operated mitral stenosis with insertion of a mechanical heart valve and CAD respectively). Twelve patients had Wolff-Parkinson-White syndrome and another had undergone surgical ablation of an accessory pathway. At the time of electrophysiologic testing, ATP was administered during episodes of paroxysmal supraventricular tachycardia, via a central vein, in incremental doses of 5, 10 and 20 mg followed by a flush of 10 c.c. of isotonic saline. The mechanism of the arrhythmia was orthodromic AV reentrant tachycardia in 19 (79%), AV nodal reentrant tachycardia in 4 (16.6%) and atrial tachycardia in one patient. The mean frequency of the tachycardia was 174 +/- 33 b.p.m. A dose of 5 mg was effective in 16 patients (66%), 5 required 10 mg and two required 20 mg for termination of the tachycardia. In the patient with atrial tachycardia ATP was not effective. The average time after injection to termination of the arrhythmia was 16 +/- 8 seconds. Orthodromic AV reentrant tachycardia was interrupted in the AV node limb in all but one patient and AV nodal reentry was terminated in the "slow-pathway" in three of the four patients. Nine patients had premature ventricular complexes, isolated or in couplets, after the termination of the SVT. Three patients had immediate recurrence of the SVT.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Efficacy and safety of adenosine triphosphate in the control of supraventricular paroxysmal tachycardia]. 804 86

Carbamoyl phosphate is the product of carbamoyl phosphate synthetase (CPS II) activity and the substrate of the aspartate transcarbamoylase (ATCase) activity, each of which is found in CAD, a large 240-kDa multienzyme polypeptide in mammals that catalyses the first three steps in pyrimidine biosynthesis. In our study of the transfer of the labile intermediate between the two active sites, we have used assays that differentiate the synthesis of carbamoyl phosphate from the overall reaction of CPS II and ATCase that produces carbamoyl aspartate. We provided excess exogenous carbamoyl phosphate and monitored its access to the respective active sites through the production of carbamoyl phosphate and carbamoyl aspartate from radiolabelled bicarbonate. Three features indicate interactions between the folded CPS II and ATCase domains causing reciprocal conformational changes. First, even in the presence of approximately 1 mM unlabelled carbamoyl phosphate, when the aspartate concentration is high ATCase uses endogenous carbamoyl phosphate for the synthesis of radiolabelled carbamoyl aspartate. In contrast, the isolated CPS II forward reaction is inhibited by excess unlabelled carbamoyl phosphate. Secondly, the affinity of the ATCase for carbamoyl phosphate and aspartate is modulated when substrates bind to CPS II. Thirdly, the transition-state analogue phosphonacetyl-L-aspartate is a less efficient inhibitor of the ATCase when the substrates for CPS II are present. All these effects operate when CPS II is in the more active P state, which is induced by high concentrations of ATP and magnesium ions and when 5'-phosphoribosyl diphosphate (the allosteric activator) is present with low concentrations of ATP; these are conditions that would be met during active biosynthesis in the cell. We propose a phenomenon of reciprocal allostery that encourages the efficient transfer of the labile intermediate within the multienzyme polypeptide CAD. In this model, binding of aspartate to the active site of ATCase causes a conformational change at the active site of the liganded form of CPS II, which protects it from inhibition by its product, carbamoyl phosphate; reciprocally, the substrates for CPS II affect the active site of ATCase by increasing the affinity for its substrates, endogenous carbamoyl phosphate and aspartate, and thus impede access of exogenous carbamoyl phosphate or the transition-state analogue. Reciprocal allostery justifies the close association of the enzyme activities within the polypeptide and ensures that carbamoyl phosphate is efficiently synthesised and is dedicated to the second step of pyrimidine biosynthesis. These conditions fulfill those required for metabolic channeling in the cell.
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PMID:A reciprocal allosteric mechanism for efficient transfer of labile intermediates between active sites in CAD, the mammalian pyrimidine-biosynthetic multienzyme polypeptide. 928 32

The synthesis of carbamoyl phosphate by the mammalian multifunctional protein, CAD, involves the concerted action of the 40 kDa amidotransferase domain (GLN), that hydrolyzes glutamine and the 120 kDa synthetase (CPS) domain that uses the ammonia, thus produced, ATP and bicarbonate to make carbamoyl phosphate. The separately cloned GLN domain has very low activity due to a reduction in kcat and an increase in Km but forms a hybrid complex with the isolated Escherichia coli CPS subunit. The hybrid has full glutamine-dependent catalytic activity and a functional interdomain linkage. The mammalian-E. coli hybrid was used to investigate the functional consequence of replacing His336 and Glu338, two residues postulated to participate in catalysis as part of a catalytic triad. The mutant mammalian GLN domains formed stable complexes with the E. coli CPS subunit, but the catalytic activity was severely impaired. While the His336Asn mutant does not form measurable amounts of the gamma-glutamyl thioester, the steady state concentration of the intermediate with the Glu338Gly mutant was comparable to the wild type hybrid because both the rate of formation and breakdown of the thioester are reduced. This result is consistent with the postulated role of Glu338 in maintaining His336 in the optimal orientation for catalysis and suggests a mechanism for the GLN CPS functional linkage.
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PMID:The function of Glu338 in the catalytic triad of the carbamoyl phosphate synthetase amidotransferase domain. 985 83

In animals, UTP feedback inhibition of carbamyl phosphate synthetase II (CPSase) controls pyrimidine biosynthesis. Suppressor of black (Su(b) or rSu(b)) mutants of Drosophila melanogaster have elevated pyrimidine pools, and this mutation has been mapped to the rudimentary locus. We report that rSu(b) is a missense mutation resulting in a glutamate to lysine substitution within the second ATP binding site (i.e. CPS.B2 domain) of CPSase. This residue corresponds to Glu780 in the Escherichia coli enzyme (Glu1153 in hamster CAD) and is universally conserved among CPSases. When a transgene expressing the Glu-->Lys substitution was introduced into Drosophila lines homozygous for the black mutation, the resulting flies exhibited the Su(b) phenotype. Partially purified CPSase from rSu(b) and transgenic flies carrying this substitution exhibited a dramatic reduction in UTP feedback inhibition. The slight UTP inhibition observed with the Su(b) enzyme in vitro was due mainly to chelation of Mg2+ by UTP. However, the Km values for glutamate, bicarbonate, and ATP obtained from the Su(b) enzyme were not significantly different from wild-type values. From these experiments, we conclude that this residue plays an essential role in the UTP allosteric response, probably in propagating the response between the effector binding site and the ATP binding site. This is the first CPSase mutation found to abolish feedback inhibition without significantly affecting other enzyme catalytic parameters.
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PMID:A mutation that uncouples allosteric regulation of carbamyl phosphate synthetase in Drosophila. 1008 Aug 91

The aim of the work was to study the O2-transport changes to tissues in cardio-surgical patients suffering from CAD and operated during extra-corporeal blood circulation (ECC). The changes of selected haematologic variables, 2,3-diphosphoglycerate (2,3-DPG) and ATP concentration, acid-base balance parameters with p50 calculation were measured in the venous blood samples taken before the operation, during the operation and on the 1st, 2nd, 3d, 5th, 7th and 10th day after the operation. From the obtained results follows, that extreme haemodilution causes significant decrease of the haematocrit value (Htc) by 35%, the value of haemoglobin (Hb) by 37% and the count of erythrocytes (Er) by 37% from the initial values. The count of reticulocytes (Ret) was increased by 52%. In the days after operation the increase in Htc values, the values of Hb and count of erythrocytes was observed, whereby the initial values were not reach even on the 10th day after the operation. The increase of the reticulocytes count by 33% prevailed to the 10th day after the operation in comparison with the initial values. 2,3-DPG concentration was increased between 3d and 10th day after the operation by 30% and ATP concentration between 5th and 10th day was increased by 23% from the initial values. Hb-O2 saturation (SpO2) and pO2 were increased already during the operation, the increase prevailed until the 7th day by 27%, pO2 until the 3d day by 39% from the initial values. Calculated values p50 did not change in the course of this study--they fluctuated in range +/- 0.04 kPa from the initial value 3.55 kPa. Supposing multifactorial character of Hb oxygenation and deoxygenation process it is possible to conclude, that the determined changes of observed parameters did not significantly influence O2-transport to tissues during ECC. (Fig. 3, Ref. 12.)
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PMID:[Oxygen transport values in patients with surgery performed under extracorporeal blood circulation]. 1103 15

Katanin, a heterodimeric protein with ATP-dependent microtubule-severing activity, localizes to the centrosome in animal cells. Widespread occurrence is suspected as several species contain homologs to the katanin p60 subunit. Recently we isolated an Arabidopsis thaliana cDNA with significant identity to the p60 subunit of sea urchin katanin. Like p60, the encoded protein is a member of the AAA superfamily of ATPases, containing the Walker ATP binding consensus and the signature AAA minimal consensus sequences within a single larger AAA/CAD amino acid motif. Phylogenetic analysis placed the encoded protein in the AAA subfamily of cytoskeleton-interactive proteins, where it formed a strongly supported clade with 4 other members identified as katanin p60 subunits. The clone was named AtKSS (Arabidopsis thaliana katanin-like protein small subunit). Western blots, performed using a polyclonal antibody raised against recombinant AtKSS, revealed AtKSS is present in protein extracts of all Arabidopsis organs examined. To evaluate potential interactions between AtKSS and the cytoskeleton, the intracellular localization of AtKSS was correlated with that of tubulin. AtKSS was found in perinuclear regions during interphase, surrounding the spindle poles during mitosis, but was absent from the preprophase band and phragmoplast microtubule arrays. These data support the thesis that AtKSS is an Arabidopsis homolog of the p60 subunit of katanin. Its cell cycle-dependent distribution is consistent with microtubule-severing activity, but additional studies will better define its role.
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PMID:cDNA isolation, characterization, and protein intracellular localization of a katanin-like p60 subunit from Arabidopsis thaliana. 1173 86


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