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At least 19 recordsLinked to original sources
PubMed1987

Interaction of acetyl phosphate and carbamyl phosphate with plant phosphoenolpyruvate carboxylase.

Acetyl phosphate produced an increase in the maximum velocity (Vmax. for the carboxylation of phosphoenolpyruvate catalysed by phosphoenolpyruvate carboxylase. The limiting Vmax. was 22.2 mumol X min-1 X mg-1 (185% of the value without acetyl phosphate). This compound also decreased the Km for phosphoenolpyruvate to 0.18 mM. The apparent activation constants for acetyl phosphate were 1.6 mM and 0.62 mM in the presence of 0.5 and 4 mM-phosphoenolpyruvate respectively. Carbamyl phosphate produced an increase in Vmax. and Km for phosphoenolpyruvate. The variation of Vmax./Km with carbamyl phosphate concentration could be described by a model in which this compound interacts with the carboxylase at two different types of sites: an allosteric activator site(s) and the substrate-binding site(s). Carbamyl phosphate was hydrolysed by the action of phosphoenolpyruvate carboxylase. The hydrolysis produced Pi and NH4+ in a 1:1 relationship. Values of Vmax. and Km were 0.11 +/- 0.01 mumol of Pi X min-1 X mg-1 and 1.4 +/- 0.1 mM, respectively, in the presence of 10 mM-NaHCO3. If HCO3- was not added, these values were 0.075 +/- 0.014 mumol of Pi X min-1 X mg-1 and 0.76 +/- 0.06 mM. Vmax./Km showed no variation between pH 6.5 and 8.5. The reaction required Mg2+; the activation constants were 0.77 and 0.31 mM at pH 6.5 and 8.5 respectively. Presumably, carbamyl phosphate is hydrolysed by phosphoenolpyruvate carboxylase by a reaction the mechanism of which is related to that of the carboxylation of phosphoenolpyruvate.

Bicarbonates↗
PubMed1983

Inorganic phosphate determination in the presence of a labile organic phosphate: assay for carbamyl phosphate phosphatase activity.

An assay was developed for carbamyl phosphate phosphatase (CAP phosphatase) activity. This enzyme activity is difficult to assay because of the chemical instability of carbamyl phosphate (CAP). We chose to measure CAP phosphatase activity by monitoring release of inorganic phosphate (Pi). This entailed preparing CAP with low endogeneous Pi, constructing enzyme incubation and deproteination conditions for minimal chemical hydrolysis of CAP and employing a Pi assay designed for use with labile organic phosphates. With Ehrlich ascites tumor cell homogenate, CAP phosphatase assay was linear with time and increments of protein. In developing this procedure, the most critical aspect was the definition of reliable conditions for measuring Pi. The Pi assay used is a modification of the method of Baginski, Foa, and Zak [Clin. Chim. Acta 15, 155-158 (1967)] which is insensitive to hydrolysis of organophosphates after the last reagent is added. Color development in the Pi assay is significantly inhibited by cold and by greater than 0.3 mmol of trichloroacetic acid or greater than 1.5 mumol of CAP. However, under conditions utilized for measuring CAP phosphatase activity there is no interference. Absorbance in the Pi assay is stable and linear from 5 to 300 nmol of Pi. The assay is also suitable for measurement of ATPase activity and Pi determinations in general.

Acid Anhydride Hydrolases↗
PubMed1976

Aspartate transcarbamylase of Escherichia coli. Heterogeneity of binding sites for carbamyl phosphate and fluorinated analogs of carbamyl phosphate.

Some preparations of both native aspartate transcarbamylase from Escherichia coli and catalytic subunit have fewer tight binding sites per oligomer for carbamyl-P than the number of catalytic peptide chains. In contrast, the number of sites for the tight-binding inhibitor N-(phosphonacetyl)-L-aspartate does equal the number of catalytic chains in each case. Binding of the labile carbamyl-P was determined using rapid gel filtration, with conversion to stable carbamyl-L-aspartate during collection. Native enzyme (six catalytic chains) obtained from cells grown under the conditions of J.C. Gerhart and H. Holoubek (J. Biol. Chem. (1967) 242, 2886-2892) has 5.4 tight sites for carbamyl-P at pH 8.0 (KD = 9.9 muM), whereas native enzyme from cells grown with higher concentrations of glucose, uracil, and histidine (to yield more enzyme per unit volume of culture) has only 1.9 tight sites at pH 8.0 (KD = 4.6 muM) and only 2.3 tight sites at pH 7.0 (KD = 2.6 muM). At pH 8.0, catalytic subunit (three catalytic chains) obtained from the former native enzyme has 2.2 tight sites for carbamyl-P (KD = 2.4 muM) and the number of sites is 2.3 in the presence of 35 mM succinate, whereas catalytic subunit obtained from the latter native enzyme has 1.8 tight sites (KD = 3.6 muM) in the absence of succinate and 2.3 tight sites in its presence. The number of tight binding sites is also less than the number of subunit peptide chains in 19F nuclear magnetic resonance experiments performed with catalytic subunit and two fluorinated analogs of carbamyl-P at comparable concentrations of analogs and active sites. A model is proposed in which incomplete removal of formylmethionine from the NH2 termini of the enzyme under conditions of extreme depression affects affinity for ligands.

Binding Sites↗
PubMed1986

Catalytic domains of carbamyl phosphate synthetase. Glutamine-hydrolyzing site of Escherichia coli carbamyl phosphate synthetase.

We present evidence that cysteine 269 of the small subunit of Escherichia coli carbamyl phosphate synthetase is essential for the hydrolysis of glutamine. When cysteine 269 is replaced with glycine or with serine by site-directed mutagenesis of the carA gene, the resulting enzymes are unable to catalyze carbamyl phosphate synthesis with glutamine as nitrogen donor. Even though the glycine 269, and particularly the serine 269 enzyme bind significant amounts of glutamine, neither glycine 269 nor serine 269 can hydrolyze glutamine. The mutations at cysteine 269 do not affect carbamyl phosphate synthesis with NH3 as substrate. The NH3-dependent activity of the mutant enzymes was equal to that of wild-type. Measurements of Km indicate that the enzyme uses unionized NH3 rather than ammonium ion as substrate. The apparent Km for NH3 of the wild-type enzyme is calculated to be about 5 mM, independent of pH. The substitution of cysteine 269 with glycine or with serine results in a decrease of the apparent Km value for NH3 from 5 mM with the wild-type to 3.9 mM with the glycine, and 2.9 mM with the serine enzyme. Neither the glycine nor the serine mutation at position 269 affects the ability of the enzyme to catalyze ATP synthesis from ADP and carbamyl phosphate. Allosteric properties of the large subunit are also unaffected. However, substitution of cysteine 269 with glycine or with serine causes an 8- and 18-fold stimulation of HCO-3 -dependent ATPase activity, respectively. The increase in ATPase activity and the decrease in apparent Km for NH3 provide additional evidence for an interaction of the glutamine binding domain of the small subunit with one of the two known ATP sites of the large subunit.

Adenosine Diphosphate↗
PubMed1987

In vivo synthesis of carbamyl phosphate from NH3 by the large subunit of Escherichia coli carbamyl phosphate synthetase.

The cloned carAB operon of Escherichia coli coding for the small and large subunits of carbamyl phosphate synthetase has been used to construct a recombinant plasmid with a 4.16 kilobase ClaI fragment of the car operon that lacks the major promoters, P1 and P2. The plasmid, pHN12, carries a functional carB gene. A mutant E. coli strain lacking both subunits of carbamyl phosphate synthetase when transformed with pHN12 overproduces the large subunit by 200-fold (8-10% of the cellular protein). The elevated levels of the large subunit enable the transformed cells to utilize NH3 but not glutamine as nitrogen donor for carbamyl phosphate synthesis. The large subunit has been purified from the overexpressing strain. The purified native large subunit is capable of synthesizing carbamyl phosphate from ammonia, HCO-3, and ATP. The kinetic properties of the large subunit compared with the holoenzyme indicate that the Michaelis constants of the large subunit for HCO-3 and ATP are modulated by its association with the small glutamine binding subunit.

Adenosine Triphosphate↗
PubMed1983

Yeast carbamyl phosphate synthetase. Structure of the yeast gene and homology to Escherichia coli carbamyl phosphate synthetase.

A cloned fragment of yeast chromosomal DNA carrying the gene CPA2 coding for the large subunit of arginine-specific carbamyl phosphate synthetase has been sequenced. The cloned DNA has a 3,354-nucleotide long continuous reading frame coding for a polypeptide of 1,117 amino acids. The calculated molecular weight of the encoded polypeptide is 123,787, in good agreement with the reported molecular weight of the yeast carbamyl phosphate synthetase large subunit. The amino acid sequence of yeast carbamyl phosphate synthetase is homologous to the recently determined sequence of Escherichia coli carbamyl phosphate synthetase (Nyunoya, H., and Lusty, C. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4629-4633) over almost the entire length of the protein. Like the E. coli large subunit, the yeast enzyme exhibits an extensive internal homology between its NH2- and carboxyl-terminal halves. The internal homology in both the yeast and E. coli proteins indicates that the gene coding for the large subunit of carbamyl phosphate synthetase was derived from a tandem duplication which occurred prior to the divergence of eukaryotes and prokaryotes.

Amino Acid Sequence↗
PubMed1990

Immunochemical analysis of carbamyl phosphate synthetase I and ornithine transcarbamylase deficient livers: elevated N-acetylglutamate level in a liver lacking carbamyl phosphate synthetase protein.

Liver tissue from 3 patients with carbamyl phosphate synthetase I deficiency and 11 patients with ornithine transcarbamylase deficiency was analyzed by Western blots for the presence of carbamyl phosphate synthetase I and ornithine transcarbamylase cross-reactive material with anti-rat enzyme antibodies. One patient with carbamyl phosphate synthetase I deficiency had no cross-reactive material with the antibody to this enzyme, one had reduced amounts, and one had similar to normal amounts. Only one of the patients with ornithine transcarbamylase deficiency had apparently normal amounts of enzyme protein in his liver, another patient had markedly reduced amounts, and the other nine patients had traces or absence of reactive ornithine transcarbamylase protein. N-acetylglutamate content in the carbamyl phosphate synthetase I deficient liver with absent enzyme protein, was higher (98.4 nmol/g) than that measured in 5 normal controls (41.6 +/- 19.3 mean +/- SD, n = 5) and much higher than the N-acetylglutamate level (2.2 nmol/g) in another carbamyl phosphate synthetase I deficient liver with normal amounts of enzyme protein. Hepatic N-acetylglutamate content in 3 patients with complete ornithine transcarbamylase deficiency was below the normal mean but within 2 standard deviations from the mean (8.5-12.8 nmol/g) whereas it was higher (30.7 nmol/g) in another partial ornithine transcarbamylase deficient patient. Most patients with ornithine transcarbamylase deficiency have markedly reduced or undetectable enzyme protein in their liver, therefore. Western blot analysis could help to establish this diagnosis in most patients. Absent carbamyl phosphate synthetase I enzyme protein may have caused elevation of the hepatic content of its cofactor, N-acetylglutamate, by an as yet unknown mechanism.

Adult↗
PubMed1980

The effects of ornithine on mitochondrial carbamyl phosphate synthesis.

When carbamyl phosphate is synthesized by isolated liver mitochondria in the absence of ornithine, the following is observed. 1. Carbamyl phosphate synthesis is progressively inhibited during the first 2 min of incubation, after which it reaches a steady rate which is 8% of that observed with ornithine. 2. Within 2 min, carbamyl phosphate accumulates in the matrix to very high levels (16 nmol/microliter) which are inhibitory to carbamyl phosphate synthetase; afterwards the levels decline, and by 15 min they are essentially the same as those found in the presence of ornithine (3 to 4 nmol/microliter). 3. The decrease in matrix carbamyl phosphate is the result of decreased synthesis and of leakage from mitochondria; 90% of the total carbamyl phosphate is in the medium after 10 min. 4. Addition of ornithine after 5 to 15 min results in rates of carbamyl phosphate synthesis essentially identical with those found when ornithine is added at the start of the incubation. 5. ATP synthesis appears to be somewhat inhibited. 6. When the accumulation of carbamyl phosphate in the medium is prevented by converting that compound to carbamyl aspartate, the total synthesis of carbamyl phosphate is not increased. Three factors appears to be involved in the low capacity of mitochondria to synthesize carbamyl phosphate in the absence of ornithine: a. inhibition of carbamyl phosphate synthetase by matrix carbamyl phosphate early in incubations; b. slight inhibition of ATP synthesis throughout the incubations; c. quantitatively most important, a very low activity of carbamyl phosphate synthetase even when matrix carbamyl phosphate is low and ATP is not limiting.

Animals↗
PubMed1975

Reversible reaction of cyanate with a reactive sulfhydryl group at the glutamine binding site of carbamyl phosphate synthetase.

Carbamyl phosphate synthetase from Escherichia coli reacts stoichiometrically (one to one) with [14C]cyanate to give a 14C-labeled complex which can be isolated by gel filtration. The formation of the complex is prevented if L-glutamine is present or if the enzyme is first reacted with 2-amino-4-oxo-5-chloropentanoic acid, a chloro ketone analog of glutamine which has been shown to react with a specific SH group in the glutamine binding site. The rate of complex formation is increased by ADP and decreased by ATP and HCO3-. The isolated complex is inactive with respect to glutamine-dependent synthetase activity. However, the reaction of cyanate with the enzyme is reversible. The rate of dissociation of the isolated complex is not affected by pH (over the pH range 6-10), is greatly increased by ATP and HCO3-, and is decreased by ADP. The allosteric effectors ornithine and UMP have no effect on either the rate of formation or the rate of dissociation of the complex; however, the apparent affinity of the enzyme for ATP is decreased by UMP and increased by ornithine. The site of reaction of cyanate with carbamyl phosphate synthetase, which is composed of a light and a heavy subunit, is with an SH group in the light subunit to give an S-carbamylcysteine residue. The binding of L-[14C]glutamine to the enzyme and the inhibition of glutamine-dependent synthetase activity by the chloroketone analog are both prevented by the presence of cyanate. The reaction with cyanate is considered to be with the same essential SH group which is located in the glutamine binding site and is alkylated by 2-amino-4-oxo-5-chloropentanoic acid. The bicarbonate-dependent effects of ATP suggest that formation of the activated carbon dioxide intermediate is accompanied by changes in the heavy subunit which functionally alter the properties of the glutamine binding site on the light subunit. The allosteric effects of ornithine and UMP are probably not related to this intersubunit interaction.

Adenosine Triphosphate↗
PubMed1986

Comparative reactivity of carbamyl phosphate and glucose 6-phosphate with the glucose-6-phosphatase of intact microsomes.

The ability of glucose 6-phosphate and carbamyl phosphate to serve as substrates for glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9) of intact and disrupted microsomes from rat liver was compared at pH 7.0. Results support carbamyl phosphate and glucose 6-phosphate as effective substrates with both. Km values for carbamyl phosphate and glucose 6-phosphate were greater with intact than with disrupted microsomes, but Vmax values were higher with the latter. The substrate translocase-catalytic unit concept of glucose-6-phosphatase function is thus confirmed. The Km values for 3-O-methyl-D-glucose and D-glucose were larger when determined with intact than with disrupted microsomes. This observation is consistent with the involvement of a translocase specific for hexose substrate as a rate-influencing determinant in phosphotransferase activity of glucose-6-phosphatase.

3-O-Methylglucose↗
PubMed2004

Environmentally determined genetic expression: clinical correlates with molecular variants of carbamyl phosphate synthetase I.

Carbamyl phosphate synthetase I (CPSI) determines the rate-limiting entry of free ammonia into the urea cycle. Disruption of CPSI affects the liver's ability to remove waste nitrogen and produce arginine, citrulline, and urea. Arginine is the necessary precursor for the critical biomolecule, nitric oxide (NO). We have studied the classic model of CPSI deficiency, which results in severe hyperammonemia, and identified a large number of molecular defects. A number of CPSI polymorphisms have been found that appear to result in functional consequences. We have examined the association of these polymorphisms with various environmental stress conditions and found that certain CPSI alleles are associated with clinical outcome. We refer to these associations as environmentally determined genetic expression (EDGE) affects. In addition to studies of classic CPSI deficiency, we have developed data for the EDGE concept in post-cardiac surgery-related pulmonary hypertension, hepatic veno-occlusive disease after bone marrow transplantation, and persistent pulmonary hypertension of the newborn. We have linked these outcomes and genotypes to the availability of the urea cycle intermediates, citrulline and arginine, and their role in NO synthesis. We hypothesize that these polymorphisms affect the functional efficiency of CPSI and thus the entire urea cycle and as such, the availability of the NO substrates. By piecing together the various functional aspects of the urea cycle changes we have seen, we can better understand the clinical vulnerabilities of patients in environmentally stressful situations. This knowledge should allow us to design intervention strategies to either predict or modify the associated adverse outcomes.

Bone Marrow Transplantation↗
PubMed1984

Characterization and significance of carbamyl phosphate phosphatase.

Carbamyl phosphate (CAP) is a key compound of the biosynthetic pathways for pyrimidines and urea. CAP can also be catabolyzed by phosphatases which hydrolyze CAP to carbamate and orthophosphate. This CAP phosphatase activity was studied from tissue preparations of Ehrlich ascites carcinoma cells, SV-40-transformed Syrian hamster cells, and normal tissues of the rat. A procedure for subcellular fractionation of Ehrlich ascites carcinoma cells was developed, so that cellular contents might be divided into nuclear, mitochondrial, lysosomal, microsomal, and cytosolic fractions. Plasma membranes were found primarily in the lysosomal fraction. There were at least 3 different CAP-hydrolyzing phosphatases, each occurring predominantly in different fractions, namely, the lysosomal, cytosolic, and microsomal fractions. These fractions contained 51, 20, and 14% of homogenate CAP phosphatase activity, respectively. The pH maximum for each of these phosphatases was 8, 5.7, and 7.5, respectively, and all fractions were stimulated by Mg2+. It was determined that activity in the lysosomal fraction resided with the plasma membrane fragments. The CAP phosphatase activities associated with the plasma membrane and cytosol were further studied. The Km for CAP of both fractions was 1.1 mM. The lysosomal fraction specific activity for CAP hydrolysis was 602 nmol/min/mg at pH 7.4 and 37 degrees and was 8-fold greater than was the specific activity of the cytosolic fraction. The two fractions differed in response to Mg2+, K+, and other ions and inhibitors. A crude subcellular fractionation of rat liver was also performed, and 92% of the activity was found in the particulate fractions. Levels of CAP phosphatase activity in homogenates of normal rat tissues varied: whole blood less than muscle less than liver less than kidney less than brain. Ehrlich ascites carcinoma cell and muscle homogenate activities were comparable at 72 and 73 nmol/min/mg at pH 7.4 and 24 degrees. CAP phosphatase activity was measured in homogenates of SV40-transformed Syrian hamster mutant cell lines with various levels of aspartate transcarbamylase and variable sensitivity to the aspartate transcarbamylase competitive inhibitor, N-(phosphonacetyl)-L-aspartate.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Anhydride Hydrolases↗
PubMed1994

Carbamyl phosphate synthetase III, an evolutionary intermediate in the transition between glutamine-dependent and ammonia-dependent carbamyl phosphate synthetases.

The amino acid sequence of carbamyl phosphate synthetase (CPS) III from liver of spiny dogfish shark Squalus acanthias was deduced from the nucleotide sequence of its cDNA. Alignment of the derived amino acid sequence of CPS III with sequences of rat and frog CPS I and hamster CPS II reveals a high degree of amino acid identity, indicating that CPS III shares the same common ancestral genes as CPSs I and II. All of the CPSs examined show a high conservation of sequences in the adenine nucleotide binding domains and in residues that have been implicated in catalysis. The active-site cysteine residue required for glutamine-dependent activity by CPS II is preserved in the sequence of CPS III. Nevertheless, analysis of the protein sequences indicates that CPS III is more closely related to CPS I than to CPS II. The structure of CPS III, which is composed of a single polypeptide, is consistent with the view that CPS III evolved by fusion of separate genes coding for the glutaminase and synthetase domains of the enzyme and, like other CPSs, the synthetase domain evolved by duplication and fusion of an ancestral kinase gene. These results, together with the recent finding that frog CPS I retains the active site cysteine residue in the glutaminase domain required for glutamine-dependent activity, indicate that other amino acid substitutions critical for glutamine-dependent activity preceded loss of this catalytic cysteine residue. The results described here together with earlier biochemical evidence support the view that acetylglutamate and glutamine-dependent CPS III found in invertebrates and fish species represents an intermediate in the evolution of ancestral glutamine-dependent CPS II toward the acetylglutamate and ammonia-dependent CPS I of ureotelic terrestrial vertebrates.

Amino Acid Sequence↗