Phosphate Synthetase

The fresh tissue is incubated in a COa/bicarbonate buffer, with ammonium bicarbonate, in the presence of ATP. The carbamyl phosphate formed reacts with added ornithine in the presence of ornithine transcarbamylase in the homogenate, to form citrulline, which is measured. 

The liver is homogenized in a solution which contains ATP (final concentration 0.01 M) and MgS04 7H20, (final concentration 0.01 Af) adjusted to pH 6.7 at 37°C with solid KHCO3. This homogenate is diluted 1 1 with cetyl ammonium bromide (CTAB) a final liver concentration of 1 in 10 is achieved, and the preparation is kept cold imtil used. 

Two solutions are required to form the substrate mixture. Solution A contains 0.03 ATP and 0.03 M MgS04 7H20, adjusted to pH 6.7 with solid KHCO3. Solution B contains 0.04 M L-omithine hydrochloride and 0.04 ilf A -acetylglutamic acid and is adjusted to pH 6.0 with approximately 1 N KOH solution, made up to 0.4 M with the calculated amount of solid ammonium bicarbonate and adjusted to pH 7.5 with solid KHCO3. The substrate is prepared by mixing 2 parts of solution A with 1 part of solution B pH about 7. It is gassed with CO2 just before use until the pH is about 6.8 at 37°. 

For the assay, 0.1 ml of homogenate is mixed with 0.3 ml of substrate solution in a small tube, and incubated at 38° for 20 minutes, taking 0.05-ml samples into 0.1 ml of 7% perchloric acid at 0, 6, 10, 15, and 20 minutes. After centrifugation, 0.05 ml of the supernatant is taken into a conical glass centrifuge tube, 0.05 ml of a 1% solution of dimethyl glyoxime in 96% ethanol added followed by 0.5 ml of an acid mixture made by dissolving 4 g of phenazone in a mixture of 76 ml of concentrated sulfuric acid, 11 ml of concentrated phosphoric acid and 163 ml 

---

Owing to the weak hydrophobicity of the PEO stationary phases and reversibility of the protein adsorption, some advantages of these columns could be expected for the isolation of labile and high-molecular weight biopolymers. Miller et al. [61] found that labile mitochondrial matrix enzymes — ornitine trans-carbomoylase and carbomoyl phosphate synthetase (M = 165 kDa) could be efficiently isolated by means of hydrophobic interaction chromatography from the crude extract. 

Martinez-Ramon, A., Knecht, E., Rubio, V., and Grisolia, S. (1990) Levels of carbamoyl phosphate synthetase I in livers of young and old rats assessed by activity and immunoassays and by electron microscopic immunogold procedures. J. Histochem. Cytochem. 38, 371-376. 

From ammonia through carbamoyl phosphate synthetase To urea... 

Diagnosis of CPS or OTC deficiency may not be apparent from the blood aminogram. Ornithine levels typically are normal. The presence of hyperammonemia, hyperglu-taminemia, hyperalaninemia and orotic aciduria in a critically ill infant affords presumptive evidence for OTC deficiency. The presence of this blood aminogram without orotic aciduria suggests carbamyl phosphate synthetase deficiency. 

Carbamyl phosphate synthetase deficiency. Carbamyl phosphate synthetase deficiency is rare. Neonates quickly develop lethargy, hypothermia, vomiting and irritability. The hyperammonemia typically is severe, even exceeding 1 mmol/1. Occasional patients with a partial enzyme deficiency have had a relapsing syndrome of lethargy and irritability upon exposure to protein. Brain damage can occur in both neonatal and late-onset groups. 

CPS carbamyl phosphate synthetase FSH follicle-stimulating hormone... 

Compound 75 served as a model for a study of the TBA reactivity of 5-O-substituted KDO derivatives17 (compare Section 11,1). A synthesis of KDO 8-phosphate (2), which occurs in Nature as the product of the KDO 8-phosphate synthetase reaction9 (Scheme 1 compare Section V,2 and 3), was performed by Charon and Szabo.113 2-O-Benzyl-D-... 

The reaction catalyzed by KDO 8-phosphate synthetase (reaction 2, Scheme 35) was first observed by Levin and Racker9 in extracts from Pseudomonas aeruginosa (see Scheme 1), and later by Ghalambor and Heath29 in extracts from Escherichia coli 0111 B4 and J-5. In the initial experiments of Levin and Racker,135 the fate of D-ribose 5-phosphate in crude bacterial extracts was studied, and the KDO 8-phosphate discovered by the authors is really derived from D-ribose 5-phosphate by three, sequential, enzyme-catalyzed reactions (see Scheme 36). 

These circumstances became apparent to the authors when they attempted to study the formation of KDO 8-phosphate as catalyzed by purified bacterial extracts. These extracts did not catalyze the formation of KDO 8-phosphate from D-ribose 5-phosphate, but required D-arabinose 5-phosphate as the substrate Heath and Ghalambor29 showed that the KDO 8-phosphate synthetase reaction, observed in Pseudomonas extracts by Levin and Racker, is also catalyzed by extracts from Escherichia coli strains 0 111 B4 and J-5. Rick and Osborn136 showed that the KDO 8-phosphate synthetase from a Salmonella typhimurium mutant conditionally defective in cell-wall synthesis had a KM of 6 mM as compared to a KM of 170 pM for the enzyme from wild-type cells. 

A comprehensive study of KDO 8-phosphate synthetase has been reported by Ray.137 The author purified the enzyme 450-fold from crude extracts of Escherichia coli B cells. The synthetase has a molecular mass of 90,000 6,000 daltons and is composed of three identical subunits having an apparent molecular mass of32,000 4,000 daltons. Two pH optima were observed, one being at pH 4.0-6.0 in succinate buffer, and the other, at pH 9.0 in glycine buffer. The isoelectric point of the enzyme is 5.1. The enzyme has an apparent KM for D-arabinose 5-phosphate of 20 pM and an apparent KM for enolpyruvate phosphate of 6 pM. 

Salmonella typhimurium mutant.136-149 The defect of this mutant lies in the apparent KM (D-arabinose 5-phosphate) of its KDO 8-phosphate synthetase (compare this Section, 2). This KM increases more than 25-fold between 29 and 42°, so that the cells become increasingly dependent on exogenously supplied D-arabinose 5-phosphate as the growth temperature is raised. Cessation of LPS biosynthesis under nonper-missive conditions is accompanied by the accumulation of a KDO-de-ficient, precursor molecule.149 Lehmann150 and Rick and coworkers151 described studies directed at the isolation and chemical characterization of such lipid A precursors (for example, 133, Scheme 39). The... 

Several observations regarding this aspect have been published, and are briefly mentioned here. 5,6-Dideoxy-6-C-phosphono-D-arabino-hexofuranose (135), an isosteric phosphonate analog of D-arabinose 5-phosphate, is apparently converted, in the presence of enolpyruvate phosphate, into 3,8,9-trideoxy-9-C-phosphono-D-mcmno-2-nonulosonic acid (136) under catalysis by KDO 8-phosphate synthetase from Escherichia coli K 235. Compound 136, an isosteric phosphonate analog of KDO 8-phosphate, is a product inhibitor of the synthetase, and, by the nature of the phosphonate group, is not subject to dephosphorylation as catalyzed by KDO 8-phosphate phosphatase156 (see Scheme 40). Compound 119 (see Scheme 33) is a weak inhibitor of KDO 8-phosphate synthetase.81 KDO inhibits KDO 8-phosphate phosphatase,139 and D-ribose 5-phosphate has an inhibitory... 

A. Pi/ erard, Control of the activity of Escherichia coli carbamoyl phosphate synthetase by antagonistic allosteric effectors. Science 154, 1572 1573 (1966). 

Urea, which contains two nitrogens, is synthesized in the liver from aspartate and carbamoyl phosphate, which in turn is produced from ammonium ion and carbon dioxide by mitochondrial carbamoyl phosphate synthetase. The urea cycle and the carbamoyl phosphate synthetase reaction are shown in Figure 1-17-2. 

Carbamoyl phosphate synthetase and ornithine transcarbamoylase are mitochondrial enzymes. 

The two conditions can be distinguished by an increase in orotic add and uracil, which occurs in ornithine transcarbamoylase deficiency, but not in the defldency of carbamoyl phosphate synthetase. Orotic acid and uracil are intermediates in pyrimidine synthrais (see Chapter 18). This pathway is stimulated by the accumulation of carbamoyl phosphate, the substrate for ornithine transcarbamoylase in the urea cycle and for aspartate transcarbamoylase in pyrimidine synthesis. 

Rate-limiting enzyme carbamoyl phosphate synthetase-1 (activated by N-acetylglutamate)... 

Carbamoyl phosphate synthetase (no increase in orotic acid or uracil)... 

Pyrimidines are synthesized de novo in the cytoplasm from aspartate, COj, and glutamine as shown in Figure 1-18-2. Synthesis involves a cytoplasmic carbamoyl phosphate synthetase that differs from the mitochondrial enzyme with the same name used in the urea cycle. 

The carbamoyl phosphate synthetase (abbreviated to CPS-I) that is involved in the ornithine cycle differs from the enzyme that is involved in pyrimidine synthesis (carbamoyl phosphate synthetase-ll). The latter enzyme is cytosolic, requires glutamine for provision of nitrogen, rather than ammonia, and is regulated by different factors (Chapter 20). 

The of carbamoyl phosphate synthetase for ammonia is assumed to be above that of the ammonia concentration in the mitochondria, so that an increase in the latter should increase the activity of the enzyme. 

Condition Carbamoyl- phosphate synthetase Ornithine transcarbamoylase Argininosuccinate synthetase Argininosuccinate lyase Arginase... 

The concentration of ammonia in the liver is not saturating for carbamoyl phosphate synthetase, so that the greater the flux of ammonia into or within the liver, the higher the concentration of ammonia and the higher the activity of the synthetase. The effect of ammonia concentration is, therefore, a mass-action effect. 

Arginine stimulates carbamoyl-phosphate synthetase (an allosteric effect). 

In view of the toxicity of ammonia, complete absence of any one of the enzymes of the cycle is fatal. Nonetheless, disorders of the cycle do occur, which are caused by a low activity of one of the enzymes or carbamoyl phosphate synthetase. In addition, defects in N-acetylglutamate synthase have been reported, but they are very rare. With the exception of ornithine transcarbamoylase, the deficiencies have an autosomal recessive mode of inheritance. The transcarbamoylase deficiency is inherited as an X-linked dominant trait, usually lethal in male patients. A deficiency of carbamoyl phosphate synthetase, ornithine transcarbamoylase or argininosuccinate synthetase results in accumulation and excretion of citrulline. A deficiency of argininosuccinate lyase results in the accumulation and excretion of argininosuccinate and arginine (Table 10.5). The abbreviations CPSD, OTCD, ASD, ALD and AD stand, respectively, for the deficiencies of these enzymes, where D stands for deficiency. 

CPS-I carbamoyl phosphate synthetase IRS insulin-receptor substrate... 

Carbamoyl phosphate synthetase formation in liver taken from tadpoles treated with thyroxine is enhanced by the addition of orotic acid, uracil or uridine (cytosine and adenosine had no effect). The synthesis of this enzyme is not affected by these pyrimidines in untreated animals. This indicates that there is a relative pyrimidine deficiency during thyroxine-induced metamorphosis [140]. 

---