Carbamoyl-phosphate synthetase activators

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. 

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

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

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. 

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. 

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. 

D208A,D210A <13> (<13> 0.1% of wild type activity [18]) [18] E136A/E138A/E141A/K140A <13> (<13> not detrimental [18]) [18] Additional information <12> (<12> enzyme can replace in vivo carbamoyl phosphate synthetase of E. coli [17]) [17]... 

These changes in demand for urea cycle activity are met over the long term by regulation of the rates of synthesis of the four urea cycle enzymes and carbamoyl phosphate synthetase I in the liver. All five enzymes are synthesized at higher rates in starving animals and in animals on veiy-high-protein diets than in well-fed animals eating primarily carbohydrates and fats. Animals on protein-free diets produce lower levels of urea cycle enzymes. 

RGURE 18-13 Synthesis of /V-acetylglutamate and its activation of carbamoyl phosphate synthetase I. 

Other therapies are more specific to a particular enzyme deficiency. Deficiency of Ai-acetylglutamate synthase results in the absence of the normal activator of carbamoyl phosphate synthetase I (Fig. 18-13). This condition can be treated by administering carbamoyl glutamate, an analog of Af-acetylglutamate that is effective in activating carbamoyl phosphate synthetase I. 

The committed step of this pathway is the synthesis of carbamoyl phosphate from glutamine and CO2, catalyzed by carbamoyl phosphate synthetase II. This enzyme is inhibited by UTP and activated by ATP and PRPP. 

Carbamoyl phosphate synthetase, which is technically not a member of the urea cycle, catalyzes the condensation and activation of ammonia (from the oxidative deamination of glutamate by glutamate dehydrogenase Topic M2) and C02 (in the form of bicarbonate, HC03 ) to form carbamoyl phosphate. The hydrolysis of two ATP molecules makes this reaction essentially irreversible. 

First the ammonium ions must be activated through conversion to carbamoyl phosphate by the mitochondrial form of carbamoyl phosphate synthetase. 

Guy, H. I., Schmidt, B., Herve, G., and Evans, D. R. (1998). Pressure-induced dissociation of carbamoyl-phosphate synthetase domains. The catalytically active form is dimeric. J Biol Chem., 273, 14172-14178. 

The end-product of the pathway, UTP, is a potent inhibitor of carbamoyl phosphate synthetase II and the substrate, ATP, also activates this enzyme. The enzymatic activity of carbamoyl phosphate synthetase II is low relative to subsequent enzymes in the pathway (Fig. 15-15) and, under normal conditions, flux through the de novo pathway may be regulated by cellular levels of P-Rib-PP, UTP and ATP i.e., carbamoyl phosphate synthetase II catalyzes the flux-controlling step in the pathway (see Section 10.5). 

There are two multifunctional proteins in the pathway for de novo biosynthesis of pyrimidine nucleotides. A trifunctional protein, called dihydroorotate synthetase (or CAD, where the letters are the initials of the three enzymatic activities), catalyzes reactions 1, 2 and 3 of the pathway (HCC>5"- CAP— CA-asp—> DHO Fig. 15-15). The enzymatic activities of carbamoyl phosphate synthetase, aspartate transcarbamoylase and dihydroorotase, are contained in discrete globular domains of a single polypeptide chain of 243 kDa, where they are covalently connected by segments of polypeptide chain whch are susceptible to digestion by proteases such as trypsin. A bifunctional enzyme, UMP synthase, catalyzes reactions 5 and 6 of the pyrimidine pathway (orotate— OMP—> UMP Fig. 15-15). Two enzymatic activities, those of orotate phosphoribosyltransferase and OMP decarboxylase, are contained in a single protein of 51.5 kDa which associates as a dimer. 

For dihydroorotate synthetase, the product of reaction 1, carbamoyl phosphate (CAP) is very unstable but is rapidly transformed by aspartate transcarbamoylase which is 50 times more active (per active site) than carbamoyl phosphate synthetase. High levels of carbamoyl aspartate (CA-asp) may be toxic, but this intermediate is rapidly consumed by the high dihydroorotase activity. Because the first three reactions are catalyzed by a single protein, the three enzyme active sites are expressed in a constant ratio under all conditions of growth this maintains CAP and CA-asp at low levels. For UMP synthase, OMP decarboxylase is far more active (per active site) than orotate PRTase, resulting in low cellular levels of the intermediate, OMP, which would otherwise be subject to enzymatic hydrolysis (in cells from higher animals). 

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