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)

Brief exposure to 6-azauridine stimulates the production of carbamoyl phosphate for de novo pyrimidine biosynthesis in vitro in slices of haematopoietic spleen from anaemic mice (preceding paper). In studies of the underlying mechanism for this response we turned our attention to changes in the level of substrates and effectors for carbamoyl-phosphate synthetase II. Intermediates of the orotic acid pathway and 6-azauridine had little effect on the synthetase activity in vitro. 6-Azauridine 5'-monophosphate (6-AzaUMP) stimulated synthetase II, possibly in an allosteric manner. However, in view of the potency as an activator and the tissue levels, 6-azaUMP may be only partially responsible for the stimulation. Adenine nucleotide levels in the tissue showed only minor changes after brief exposure (15 min) to 6-azauridine. The level of UTP and UDP, potent inhibitors for synthetase II, showed no significant change. The level of 5-phosphoribosyl 1-pyrophosphate (PPRibP), a potent positive effector for the synthetase II, showed a more than 1.5-fold increase after 15 min. The relative importance of these factors was evaluated by assay of the synthetase, partially purified from mouse spleen, under simulated conditions in vitro. The results indicated that the enhanced level of PPRibP played a major role in increasing the production of carbamoyl phosphate. In Ehrlich ascites cells in vitro, where 6-azauridine did not increase carbamoyl phosphate production, the basal PPRibP level was high (range over 0.1 mM) and the changes in the level, brought about by the analogue, were relatively small.
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PMID:Enhancement of intracellular 5-phosphoribosyl 1-pyrophosphate levels as a major factor in the 6-azauridine-induced stimulation of carbamoyl phosphate synthesis in mouse spleen slices. 618 35

Carbamyl phosphate synthetase-I (CPS-I)2, purified from mouse hepatic mitochondria consists of electrophoretically homogeneous polypeptide species of 160 kilodaltons molecular weight (Kd). Monospecific antibody to CPS-I immunoprecipitated a putative precursor of 165Kd protein from in vitro translation products programmed with mouse liver free polysomes or poly(A) RNA. Isolated mitochondrial particles can take up and process pCPS-I into mature CPS-I of 160Kd in an in vitro transport system. The in vitro transport of CPS-I is energy dependent and requires intact mitochondria. The processing of pCPS-I appears to involve a single endoproteolytic cleavage.
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PMID:The transport and processing of carbamyl phosphate synthetase-I in mouse hepatic mitochondria. 620 Jan 5

The pyr-3 gene of Neurospora crassa codes for the bifunctional enzyme pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyltransferase (carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating)/carbamoylphosphate: L-aspartate carbamoyltransferase), EC 6.3.4.16/EC 2.1.3.2). We describe the investigation of substrate- and product-binding sites of the enzyme by affinity chromatography, using the ligands aspartate, glutamate, and adenosine 5'-diphosphate, and investigate the channelling of carbamoyl phosphate, the product of the first function and substrate of the second, through the pathway. For this latter aspect of the investigation, two new enzyme assays were devised and described. The results of the competition studies on carbamoyl phosphate-binding are consistent with the existence of two different binding sites within the enzyme for this metabolic intermediate, one for it as the product of the first step and the other for it as the substrate of the second.
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PMID:Investigation of binding sites in the complex pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyltransferase enzyme of Neurospora crassa. 621 40

Rat liver carbamoyl-phosphate synthetase I is shown to have synthetase and ATPase activity in the absence of acetylglutamate. Km values for ATP, Mg2+ and K+ are greatly increased, the Km for HCO-3 is not changed much, and the Km for NH+4 is markedly reduced. Vmax for the synthetase reaction is less than 20% of that of the acetylglutamate-activated enzyme whereas Vmax for the ATPase activity is greater than 40% of that with acetylglutamate. Pulse-chase experiments with H14CO-3 show formation of less "active CO2" (the central intermediate) than with acetylglutamate; ATPase activity is reduced in proportion, but the synthetase activity is much smaller. Binding of one ATP molecule with high affinity (Kd = 20-30 microM) is shown in the absence of acetylglutamate. This appears to be the molecule of ATPB (ATPB provides the phosphoryl group of carbamoyl phosphate). In contrast, the affinity for ATPA (ATPA yields Pi) is much reduced. Initial velocity measurements without acetylglutamate show a time lag before reaching a constant velocity. At 50 microM acetylglutamate the lag is much longer, but at 10 mM acetylglutamate it is shorter. Activation by acetylglutamate requires ATP at concentrations sufficient to occupy the ATPA and the ATPB binding sites. Preincubation with 10 mM acetylglutamate alone shortens the activation time. From these findings we propose an allosteric model for activation of carbamoyl-phosphate synthetase in which there are two active states, R and R . AcGlu. Binding of ATPA is associated with the conversion of T to R. R . AcGlu differs from R in that transfer to carbamate of the gamma-phosphoryl group of ATPB appears to be facilitated.
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PMID:Mitochondrial carbamoyl phosphate synthetase activity in the absence of N-acetyl-L-glutamate. Mechanism of activation by this cofactor. 622 15

The mechanism of the reaction catalyzed by rat liver mitochondrial carbamoyl-phosphate synthetase has been studied by using [beta-18O2]ATP and HC18O-3, monitoring the isotopic composition of adenosine triphosphate (ATP) and inorganic phosphate (Pi) by high-resolution 31P NMR spectroscopy. In the presence of both HCO3- and acetylglutamate, the enzyme catalyzes the exchange of oxygen atoms between the beta, gamma bridging and the beta nonbridging positions of ATP. Addition of NH3 stops the exchange, Pi released by the ATPase activity of the enzyme in the absence of NH3 contains one oxygen atom from HC18O3- but there is no incorporation of 18O into ATP. There is no significant incorporation of [14C]ADP or 32Pi into ATP. It is concluded that in the enzyme-ATPA.HCO30.ATPB complex formed in the presence of ATP and HCO3- there is reversible transfer of the gamma-PO3 group of ATPA (the molecule that yields Pi) to HCO3- without dissociation of products. The beta-PO3 of the enzyme-bound ADP that is formed can rotate. Virtually all of the complex appears to be in the form in which ATPA is cleaved, but in the absence of NH3, ATP is reconstituted and dissociates from the complex on at least 75% of the occasions. On the remainder, the carbonyl phosphate is cleaved in an irreversible process that yields Pi and a low-energy form of carbonic acid (probably HCO3-). NH3 reacts rapidly and irreversibly with the complex, and at saturation the rate (greater than 10 times the rate of Pi release in the absence of NH3) is sufficient to prevent dissociation of ATPA. In the absence of HCO3- an enzyme-ATPA.ATPB complex is formed, but cleavage of the bond between beta, gamma bridging oxygen and P gamma of ATPA does not occur.
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PMID:Mechanism of activation of bicarbonate ion by mitochondrial carbamoyl-phosphate synthetase: formation of enzyme-bound adenosine diphosphate from the adenosine triphosphate that yields inorganic phosphate. 626 8

Carbamoyl-phosphate synthetase II (glutamine-hydrolyzing) (EC 6.3.5.5) (synthetase II), is the first and rate-limiting enzyme in the de novo UMP biosynthetic pathway. The present investigation showed that insulin has a regulatory action on hepatic synthetase II activity. When diabetes was induced with injection of different doses of alloxan the plasma insulin concentrations decreased in a dose-dependent fashion to 72, 38, 31 and 28% and concurrently the liver synthetase II activity decreased to 75, 43, 29 and 22% of the normal values. In diabetic rats dose response studies showed that with insulin injections of 4, 6, 8 or 10 U/day for 48 h the hepatic synthetase II activity increased to 81, 95, 99 and 103% of the control liver values. In the diabetic rats the insulin-induced rise in liver synthetase II activity was prevented by treatment of the rats with actinomycin.
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PMID:Action of insulin on liver carbamoyl-phosphate synthetase II (glutamine-hydrolyzing) activity. 634 26

Carbamyl phosphate synthetase-I (CPS-I; EC 6.3.4.16), a mitochondrial enzyme of the urea-cycle, was studied in deactivated extracts of rat liver. It has been found to be activated in vitro by dithiothreitol (DTT) and Mg2+ ions. After reduction by DTT, thioredoxins, isolated from rat liver, were able to activate CPS-I by 468%.
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PMID:[In vitro activation by dithiothreitol and thioredoxins of carbamyl phosphate synthetase-I in rat liver]. 644 19

Carbamyl phosphate synthetase-I (CPS-I) catalyzes the first reaction required for the conversion of ammonia to urea through the urea cycle. Severe CPS-I deficiency causes marked hyperammonemia with encephalopathy in infancy and usually results in death within the first few months of life. We describe a 33-year-old woman whose CPS-I activity is less than 5% of normal. She has had mild, intermittent symptoms throughout life but has never experienced severe encephalopathy. Although mildly retarded, she has no major neurological deficits. Therapy with a low-protein diet, lactulose, and sodium benzoate has prevented recurrence of hyperammonemia and symptoms. Cranial computed tomographic scans demonstrate prominent lucency of cerebral white matter, and cerebral evoked potential recordings indicate slowed central conduction. These findings suggest that the metabolic disturbances in this patient may have adversely affected central myelin formation or maintenance. This woman represents, to our knowledge, the oldest reported patient with CPS-I deficiency, and the case illustrates the need to consider urea cycle disorders in the differential diagnosis of intermittent neurological symptoms regardless of the patient's age.
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PMID:Clinical features of carbamyl phosphate synthetase-I deficiency in an adult. 646 66

The glutamine- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase III present in liver of largemouth bass (Micropterus salmoides) has been highly purified. The properties of the enzyme are generally similar to the properties of the carbamoyl phosphate synthetase III from spiny dogfish (Squalus acanthias) previously described (Anderson, P. M. (1981) J. Biol. Chem. 256, 12228-12238). However, the bass enzyme is not subject to self-association, and the effects of urea and, particularly, trimethylamine-N-oxide, on catalytic activity are considerably reduced. Ammonia can substitute for glutamine as the nitrogen-donating substrate, but the maximum rate is lower. Carbamoyl phosphate synthetase III, like other carbamoyl phosphate synthetases, catalyzes two partial reactions, ATP synthesis from carbamoyl phosphate and ADP, and bicarbonate-dependent hydrolysis of ATP; both reactions are greatly stimulated by the presence of N-acetyl-L-glutamate. Carbamoyl phosphate synthetase III gave no detectable immunological cross-reaction with antibody to the ammonia- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase from rat liver mitochondria. The apparent Km value for N-acetyl-L-glutamate decreases significantly as the concentration of L-glutamine increases in the glutamine-dependent reaction, and vice versa. This effect is glutamine-specific. The apparent Km for N-acetyl-L-glutamate in the ammonia-dependent reaction is not affected by changes in ammonia concentration and the apparent Km for ammonia (8 mM) is also not affected by changes in N-acetyl-L-glutamate concentration. Studies involving inhibition of carbamoyl phosphate synthetase III by the glutamine analogs acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), DON (6-diazo-5-oxo-L-norleucine), and chloroketone (L-2-amino-4-oxo-5-chloropentanoic acid), provided additional evidence for significant interaction between the L-glutamine- and N-acetyl-L-glutamate-binding sites. Glutamine-dependent but not ammonia-dependent activity is inhibited by preincubating the enzyme with these analogs. This inhibition requires the presence of both MgATP and N-acetyl-L-glutamate, and is prevented by the additional presence of L-glutamine. Inhibition of the glutamine-dependent reaction by DON or chloroketone is accompanied by a decrease in the apparent Km for N-acetyl-L-glutamate in the ammonia-dependent reaction from 0.3 mM to a value which is nearly the same as that observed in the glutamine-dependent reaction when glutamine is saturating (0.015 mM).
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PMID:Glutamine- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase from Micropterus salmoides. Purification, properties, and inhibition by glutamine analogs. 660 5

The amount of control of the various steps involved in the citrulline synthesizing pathway in isolated rat-liver mitochondria incubated with saturating concentrations of ammonia and ornithine has been measured. The flux control coefficient of carbamoyl-phosphate synthase (ammonia) was close to one. Control exerted by other steps in the pathway, including ornithine transcarbamoylase, carbonic anhydrase. ATP production and transport of ornithine, was negligible. The high flux control coefficient of carbamoyl-phosphate synthetase is due to the low elasticity coefficient of this enzyme towards its product, intramitochondrial carbamoyl phosphate, in combination with a high elasticity coefficient of ornithine transcarbamoylase towards carbamoyl phosphate.
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PMID:Analysis of the control of citrulline synthesis in isolated rat-liver mitochondria. 674 75


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