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)

Glutamine-dependent carbamoyl-phosphate synthetase was purified about 2100-fold from the cytosol of rat liver using 30% (v/v) dimethyl sulfoxide and 5% (w/v) glycerol as stabilizers. Throughout the purification, aspartate transcarbamylase and dihydroorotase, the second and third enzymes of pyrimidine biosynthesis, were copurified with the synthetase. These three enzymes sedimented as a single peak with a sedimentation coefficient of 27 S in sucrose gradients containing the stabilizers, indicating their existence as a multienzyme complex. The aggregation states of the complex were analyzed by sucrose gradient centrifugation under conditions approximating those used for enzymatic assay and correlated with the kinetic properties of the synthetase. In the presence of 10% glycerol and 10 mM MgATP(2-) at 18 degrees, the synthetase showed high activity and the three enzymes sedimented as a single peak with a coefficient of 25 S. The three enzymes also existed as a complex with the same coefficient when 50 muM PP-ribose-P was added in place of MgATP(2-), the sedimentation coefficient of the complex shifted to 28 S, indicating alteration in its molecular shape, rather than size. With 10% glycerol alone, the complex partially dissociated and the synthetase activity appeared in three peaks with coefficients of 26, 19, and 9 S (carbamoyl-phosphate synthetases (CPSase) a, b, and c, respectively). CPSases a, b, and c, thus obtained, were all sensitive to regulation by UTP and PP-ribose-P, but they differed MgATP(2-) (5.1, 4.8, AND 1.7 mM for CPSases a and b, and the enzyme within the original complex, respectively) and in their sensitivities to effectors. These results suggest that the aggregation may modify the catalytic and regulatory properties of the synthetase; Attempts to reassociate the components were unsuccessful.
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PMID:Aggregation states and catalytic properties of the multienzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver. 114 71

Acetylglutamate and ATP accelerate the oxidative inactivation of carbamoyl phosphate synthetase I by mixtures of Fe3+, ascorbate, and O2, but the mechanism of the inactivation differs with each ligand. In the presence of acetylglutamate, MgATP prevents, Mg2+, Mn2+, and catalase have no effect, and EDTA increases the inactivation, and the two phosphorylation steps of the enzyme reaction are lost simultaneously. The inactivation appears to be mediated by dehydroascorbate and is associated with the reversible oxidation of the highly reactive cysteines 1327 and 1337 and with oxidation of non-thiolic groups in the second 40-kDa domain (the enzyme consists of 4 domains of 40, 40, 60, and 20 kDa, from the amino terminus). The data are consistent with oxidation of groups at or near the site for ATPA (ATPA yields Pi; ATPB yields carbamoyl phosphate), and with the location of this site at the interphase between the second 40-kDa and the COOH-terminal domains. The oxidative inactivation promoted by ATP is inhibited by Mg2+, Mn2+, catalase, and EDTA, is not mediated by dehydroascorbate, and is not associated with oxidation of cysteines 1327 and 1337. Groups in the 60-kDa domain are oxidized. The phosphorylation step involving ATPB is lost preferentially, and the inactivation and the binding of ATPB exhibit the same dependency on the concentration of ATP. The results indicate that the oxidation is catalyzed by FeATP bound at the site for ATPB and support the binding of ATPB in the 60-kDa domain. We also demonstrate that mercaptoethanol, reducing impurities in glycerol, and dithioerythritol, in the presence of EDTA, replace ascorbate in the oxidative system. In addition, we study the influence of the oxidation on the degradation of the enzyme by rat liver lysosomes, mitochondria, and cytosol.
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PMID:Oxidative inactivation of carbamoyl phosphate synthetase (ammonia). Mechanism and sites of oxidation, degradation of the oxidized enzyme, and inactivation by glycerol, EDTA, and thiol protecting agents. 153 38

When we incubated biotin carboxylase from Escherichia coli with ATP in absence of biotin we observed HCO3- -dependent ATP hydrolysis, which was activated by 10% ethanol in the same proportion as the activity of D-biotin carboxylation assayed in the presence of biotin. The two activities exhibited identical heat stability and were protected equally by glycerol; both required Mg2+ and K+ and showed similar dependency on the concentration of ATP. Biotin assay excluded potential contamination by traces of biotin as a cause of the observed ATP hydrolysis, and this was confirmed by the findings that carboxybiotin did not accumulate and that avidin was uninhibitory. Therefore we concluded that this HCO3- -dependent ATPase was genuinely a partial activity of biotin carboxylase. This partial activity supports a sequential mechanism for enzymatic carboxylation of biotin in which HCO3- is activated by ATP in a first step. It is consistent with the initial formation of the carbonic-phosphoric anhydride (HOCO2PO3(2-)), and it does not agree with models where biotin is phosphorylated by ATP prior to reaction with HCO3-. It appears that enzymes that use HCO3- for carboxylation, including biotin-dependent carboxylases, phosphoenolpyruvate carboxylase, and carbamoyl phosphate synthetase, activate HCO3- by a common mechanism involving the initial formation of the carbonic-phosphoric anhydride.
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PMID:ATPase activity of biotin carboxylase provides evidence for initial activation of HCO3- by ATP in the carboxylation of biotin. 294 46

A carbamoyl-phosphate synthase has been purified from mycelia of Phycomyces blakesleeanus NRRL 1555 (-). The molecular weight of the enzyme was estimated to be 188,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consists of two unequal subunits with molecular weights of 130,000 and 55,000. The purified enzyme has been shown to be highly unstable. The carbamoyl-phosphate synthase from Phycomyces uses ammonia and not L-glutamine as a primary N donor and does not require activation by N-acetyl-L-glutamate, but it does require free Mg2+ for maximal activity. Kinetic studies showed a hyperbolic behavior with respect to ammonia (Km 6.34 mM), bicarbonate (Km 10.5 mM) and ATP.2 Mg2+ (Km 0.93 mM). The optimum pH of the enzyme activity was 7.4-7.8. The Phycomyces carbamoyl-phosphate synthase showed a transition temperature at 38.5 degrees C. It was completely indifferent to ornithine, cysteine, glycine, IMP, dithiothreitol, glycerol, UMP, UDP and UTP. The enzyme was inhibited by reaction with 5 mM N-ethylmaleimide.
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PMID:Carbamoyl-phosphate synthase in Phycomyces blakesleeanus. 339 22

A high specific activity of carbamoyl-phosphate synthetase II (glutamine-hydrolyzing; EC 6.3.5.5) was demonstrated in extract of the cultured Crithidia fasciculata. The enzyme was separated from aspartate carbamoyltransferase by ammonium sulfate fractionation. Apparent Km for the synthetase for L-glutamine, NH4+, MgATP or bicarbonate was 0.27, 26, 1.7 or 1.7 mM at 2.0% dimethyl sulfoxide plus 0.3% glycerol. 8.6% dimethyl sulfoxide plus 1.4% glycerol decreased Km for L-glutamine to 0.10 mM, while Km for MgATP was unaffected. The higher solvent concentrations made Vmax markedly reduced, yielding the inhibition of the activity. These properties are unique to the Crithidia synthetase, compared with the mammalian enzyme.
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PMID:Kinetic properties of carbamoyl-phosphate synthetase II (glutamine-hydrolyzing) in the parasitic protozoan Crithidia fasciculata and separation of the enzyme from aspartate carbamoyltransferase. 360 29

Carbamoyl-phosphate synthetase II of higher animals, the first enzyme of de novo pyrimidine biosynthesis, forms a multienzyme complex with aspartate carbamoyltransferase and dihydroorotase, the second and third enzymes of the pathway. The hypothesis that the complex serves to channel carbamoyl-phosphate, synthesized by the first enzyme of the complex, to the second enzyme was tested using a highly purified complex preparation from Yoshida ascites hepatoma cells (AH 13). Experimentally, aspartate carbamoyltransferase in the complex was allowed to compete with exogenously added ornithine carbamoyltransferase, another carbamoyl-phosphate-utilizing enzyme, for carbamoyl-phosphate which was either synthesized endogenously or added exogenously. The ratios of amounts of the two enzymic products, carbamoyl-aspartate and citrulline, were compared. In the absence of enzyme stabilizers dimethyl sulfoxide or glycerol, a slight channeling of the intermediate in the complex was observed. The further addition of 5-phosphoribosyl 1-pyrophosphate, MgUTP (positive and negative allosteric effectors of carbamoyl-phosphate synthetase II), 30% (v/v) dimethyl sulfoxide or 30% (w/v) glycerol did not affect the extent of channeling. It was slightly increased in the presence of 7.5% (v/v) dimethyl sulfoxide plus 2.5% (w/v) glycerol. Any shift of the assay temperature, pH or concentration of MgATP or of the enzyme complex resulted in little further increase in the extent of channeling. Even when a larger amount of the enzyme complex was used to approximate physiological conditions, there was no increase in the extent of channeling either without or with allosteric effectors. MgUTP even abolished channeling under these conditions. These results indicate that carbamoyl-phosphate can be channeled in the multienzyme complex of AH 13 cells, but the extent of channeling is very small, contrary to expectation.
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PMID:Studies on channeling of carbamoyl-phosphate in the multienzyme complex that initiates pyrimidine biosynthesis in rat ascites hepatoma cells. 613 83

The 6 enzymes involved in de novo synthesis of pyrimidines were measured in Plasmodium falciparum isolated by saponin lysis from RBC's nonsynchronized and synchronized in vitro cultures. The total activities were found to be dependent on the stage of the P. falciparum cycle. In parasites isolated from synchronized cultures, the highest activities for all enzymes were found at about 27 hr after synchronization in the late trophozoite stage, or just before schizont formation. Merozoites and ring forms contained little de novo activity. The first enzyme of the pathway, carbamyl phosphate synthetase (CPS-II) preferentially utilized glutamine. Ammonia was a poor substrate. CPS-II was unstable in the absence of the cryoprotectants, dimethylsulfoxide and glycerol. The apparent Km for MgATP--was 3.8 +/- 0.7 mM and the enzyme in all morphological forms of P. falciparum (ring, mature trophozoites and schizonts) was inhibited by UTP. The activity of the fourth enzyme of the pathway, dihydroorotate dehydrogenase, appeared to be linked to the cell's respiratory chain; inhibitors of mammalian electron transport such as cyanide, amytal, antimycin A, thenoyltrifluoroacetone and ubiquinone analogs also inhibited the P. falciparum enzyme. The demonstration of the variation of activity of the pyrimidine enzymes correlates with the increased synthesis of nucleic acids in the late trophozoite stage. These observations provide a basis for the testing of the effectiveness of pyrimidine analogs as potential antimetabolites against various forms of the parasite.
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PMID:Pyrimidine de novo synthesis during the life cycle of the intraerythrocytic stage of Plasmodium falciparum. 615 76

The pyrimidine-3 gene of Neurospora crassa codes for a bifunctional enzyme catalysing the first two steps of the pyrimidine biosynthetic pathway. Difficulties have been experienced in purification due to the lability of the enzyme. The enzyme loses carbamoyl-phosphate synthetase (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16) activity and undergoes a change in apparent molecular weight from the native 650,000 to 100,000 of the only detectable fragment. Attempts have been made therefore to stabilize the enzyme so as to minimise these effects. Elastinal, a protease inhibitor, reduces the effects, as do certain ultraviolet-sensitive mutant strains which lack a minor protease. The nature of the loss of carbamoyl-phosphate synthetase suggests an instability in the tertiary structure of the enzyme which can be reduced by the use of glycerol. Glycerol also exhibits a protease-inhibiting effect in this system. Although a range of protease inhibtors and use of uvs mutants can reduce the rate of decay of carbamoyl-phosphate synthetase activity, only glycerol can stabilize the native molecular weight. Our results support the hypothesis that the loss of carbamoyl-phosphate synthetase activity and change in molecular weight of the enzyme is a three-step sequence of proteolysis, conformational shift and cleavage of a further non-covalent bond.
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PMID:The involvement of proteolysis in conformational stability of the carbamoyl-phosphate synthetase/aspartate carbamoyltransferase enzyme of Neurospora crassa. 645 44

All six enzymes of the de novo biosynthetic pathway leading to the biosynthesis of UMP have been characterized in Toxoplasma gondii. The first three enzymes of the pathway, carbamyl phosphate synthetase-II (CPS-II), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) could be consistently separated by sucrose gradient centrifugation. Their molecular weights were estimated to be approximately 540 000, 140 000 and 70 000, respectively. The last two enzymes, orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase), cosedimented at the same position, corresponding also to a molecular weight of approximately 70 000. The fourth enzyme, dihydroorotate dehydrogenase (DHO-DHase), was associated with the particulate fraction. Apparent Km values for the respective enzymes were: CPS-II, MgATP2- (19.7 1.2 mM), L-glutamine (12.0 +/- 1.7 microM), ammonia (15.5 +/- 2.7 mM); ATCase, carbamyl phosphate (26.2 +/- 3.5 microM), L-aspartate (17.6 +/- 8.5 mM); DHOase (reverse direction) dihydroorotate (1.6 +/- 0.08 microM); ODCase, orotidine 5'-monophosphate (0.41 +/- 0.04 microM). MgUTP2- was found to act as an inhibitor of CPS-II, with an apparent Ki of 0.41 mM. However, 5-phospho-alpha-D-ribosyl-1-diphosphate, dimethyl sulphoxide and glycerol had no effect on the Km value for MgATP2-. The effect of some inhibitors, including pyrimidine and purine nucleotides and analogs and respiratory chain inhibitors, was also determined for the enzymes of the pathway.
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PMID:Enzymes of the de novo pyrimidine biosynthetic pathway in Toxoplasma gondii. 685 12

The binding of N-acetyl-L-glutamate, the physiological allosteric activator, to rat liver carbamoyl-phosphate synthetase (ammonia) was studied by techniques of rate of dialysis and of ultracentrifugation in the Airfuge. There is one binding site for acetylglutamate per enzyme monomer (Mr 165 000). K+, Mg2+ (free) and ATP were required to demonstrate binding. The concentrations of ATP required indicate that binding of ATPA (the ATP molecule that yields Pi) is needed. HCO-3 was not essential, but it enhanced binding of acetylglutamate. Glycerol also favored binding. Plots of Kd values versus the reciprocal of free Mg2+ and ATP concentrations are linear and indicate that ATPA, K+ and Mg2+ bind before acetylglutamate. In the presence of these ligands and HCO-3, ammonia increased drastically the Kd value for acetylglutamate, whereas in absence of HCO-3 ammonia had little effect. This suggests that acetylglutamate dissociates with the products and explains the higher Km for acetylglutamate in the synthetase (overall) reaction than in the ATPase (partial) reaction. In the absence of ATP acetylglutamate was bound with high affinity if ADP and carbamoyl phosphate were present. ADP or carbamoyl phosphate alone did not promote substantial binding. Binding of acetylglutamate at low concentration was slow; it was accelerated at higher concentrations of the activator. Exchange of bound acetylglutamate with acetylglutamate in solution was fast. A scheme proposed earlier for allosteric activation of the enzyme [Rubio, V., Britton, H. G. and Grisolia, S. (1983) Eur. J. Biochem. (in preparation)] is refined to incorporate the new information. Binding of ATPA, K+ and Mg2+ and formation of 'active CO2' (the central complex) are greatly favored by acetylglutamate.
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PMID:Binding of N-acetyl-L-glutamate to rat liver carbamoyl phosphate synthetase (ammonia). 688 68


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