Enzymes carbamoyl-phosphate synthase

Condensation of CO2, ammonia, and ATP to form carbamoyl phosphate is catalyzed by mitochondrial carbamoyl phosphate synthase I (reaction 1, Figure 29-9). A cytosolic form of this enzyme, carbamoyl phosphate synthase II, uses glutamine rather than ammonia as the nitrogen donor and functions in pyrimidine biosynthesis (see Chapter 34). Carbamoyl phosphate synthase I, the rate-hmiting enzyme of the urea cycle, is active only in the presence of its allosteric activator JV-acetylglutamate, which enhances the affinity of the synthase for ATP. Formation of carbamoyl phosphate requires 2 mol of ATP, one of which serves as a phosphate donor. Conversion of the second ATP to AMP and pyrophosphate, coupled to the hydrolysis of pyrophosphate to orthophosphate, provides the driving... 

The enzyme carbamoyl phosphate synthase (CPS) is a control point in the process. Stage 3. The urea cycle (Figure 6.7)... 

The mechanism of formation of carbamoyl phosphate. The reaction involves three steps, all of which take place on the same enzyme, carbamoyl phosphate synthase. 

High concentrations may cause significant respiratory failure with acute lung injury, non-cardiogenic pulmonary oedema and ARDS. Ammonia is converted to carbamoyl phosphate by the enzyme carbamoyl phosphate synthase, and then enters the urea cycle to be either incorporated into amino acids or excreted in the urine. 

Carbamoyl Phosphate Synthase I Is the Pacemaker Enzyme of the Urea Cycle... 

The activity of carbamoyl phosphate synthase I is determined by A -acetylglutamate, whose steady-state level is dictated by its rate of synthesis from acetyl-CoA and glutamate and its rate of hydrolysis to acetate and glutamate. These reactions are catalyzed by A -acetylglu-tamate synthase and A -acetylglutamate hydrolase, respectively. Major changes in diet can increase the concentrations of individual urea cycle enzymes 10-fold to 20-fold. Starvation, for example, elevates enzyme levels, presumably to cope with the increased production... 

Changes in enzyme levels and allosteric regulation of carbamoyl phosphate synthase by A -acetylglutamate regulate urea biosynthesis. 

Figure 34-7 summarizes the roles of the intermediates and enzymes of pyrimidine nucleotide biosynthesis. The catalyst for the initial reaction is cytosolic carbamoyl phosphate synthase II, a different enzyme from the mitochondrial carbamoyl phosphate synthase I of urea synthesis (Figure 29-9). Compartmentation thus provides two independent pools of carbamoyl phosphate. PRPP, an early participant in purine nucleotide synthesis (Figure 34-2), is a much later participant in pyrimidine biosynthesis.

Some of the results obtained by differential centrifugation showed enzyme distribution between different cell fractions which were difficult to interpret. Enzymes like carbamoyl phosphate synthase or isocitrate dehydrogenase were found both in mitochondria and in the soluble fraction of the cell. This led to detailed kinetic studies with purified enzymes which indicated there might be populations of enzymes with slightly different properties (isozymes) catalyzing similar reactions in different compartments or in different cell types. Variations in kinetic behavior appeared to tailor the enzyme appropriately to the particular compartment or cell where the reaction took place. 

Carbonic anhydrase (CA, also called carbonate dehydratase) is an enzyme found in most human tissues. As well as its renal role in regulating pH homeostasis (described below) CA is required in other tissues to generate bicarbonate needed as a co-substrate for carboxylase enzymes, for example pyruvate carboxylase and acetyl-CoA carboxylase, and some synthase enzymes such as carbamoyl phosphate synthases I and II. At least 12 isoenzymes of CA (CA I—XII) have been identified with molecular masses varying between 29 000 and 58 000 some isoenzymes are found free in the cytosol, others are membrane-bound and two are mitochondrial. 

The regulation of bacterial aspartate carbamoyltransferase by ATP and CTP has been particularly well studied, and is discussed on p. 116. In animals, in contrast to prokaryotes, it is not ACTase but carbamoyl-phosphate synthase that is the key enzyme in pyrimidine synthesis. It is activated by ATP and PRPP and inhibited by UTP. 

The complete urea cycle as it occurs in the mammalian liver requires five enzymes Argininosuccinate synthase, arginase, and argininosuccinate lyase (which function in the cytosol), and ornithine transcarbamoylase, and carbamoyl phosphate synthase (which function in the mitochondria). Additional specific transport proteins are required for the mitochondrial uptake of L-ornithine, NH3, and HC03 and for the release of L-citrulline. 

The pathway for UMP synthesis is shown in figure 23.13. It starts with the synthesis of carbamoyl phosphate, catalyzed by carbamoyl phosphate synthase. This enzyme is present in microorganisms and in the cytosol of all eukaryotic cells... 

Low activities of orotidine phosphate decarboxylase and (usually) orotate phosphoribosyltransferase are associated with a genetic disease in children that is characterized by abnormal growth, megaloblastic anemia, and the excretion of large amounts of orotate. When affected children are fed a pyrimidine nucleoside, usually uridine, the anemia decreases and the excretion of orotate diminishes. A likely explanation for the improvement is that the ingested uridine is phosphorylated to UMP, which is then converted to other pyrimidine nucleotides so that nucleic acid and protein synthesis can resume. In addition, the increased intracellular concentrations of pyrimidine nucleotides inhibit carbamoyl phosphate synthase, the first enzyme in the. naibwav of aro-tate synthesis. 

Carbamoyl phosphate synthase contributes to two processes (a) the initial enzyme in the biosynthesis of pyrimidines and (b) a component in the synthesis of arginine biosynthesis or the urea cycle. In bacteria both of these processes occur within the same compartment. In human beings the carbamoyl phosphate synthase involved in the urea cycle is contained in... 

The reaction is catalysed by carbamoyl phosphate syndiase which is Mg -dependent and has an absolute requirement for N-acetylglutamate as a cofactor. Carbamoyl phosphate synthase is a mitochondrial enzyme and comprises approximately 15% of the matrix protein in liver mitochondria. As a consequence of the hydrolysis of two molecules of ATP, the formation of carbamoyl phosphate is effectively irreversible, so that any ammonia undergoing this reaction is committed to excretion. 

It is of interest that many of the key enzymes in urea synthesis are located in the mitochondria. The mitochondrial enzymes include several transaminases, especially glutamate-oxaloacetate transaminase, as well as glutamate dehydrogenase, carbamoyl phosphate synthase, ornithine transcarbamylase and A -acetylglutamate synthase. 

From what has been said it is apparent that it is the different enzyme patterns which arise firom the tissue-specific expression of genes that mainly account for the varying metabolic capabilities of different tissue. Thus, for example, the enzymes fructose 1,6-bisphosphatase and glucose 6-phosphatase occur only in liver and kidney and these are the only tissues capable of gluconeogenesis. Similarly, carbamoyl phosphate synthase, which is involved in the synthesis of urea and is ammonia dependent, occurs in the mitochondria of liver but not of other tissues. At the same time, the enzymic profile of tissues can vary to some extent in response to external factors such as diet. 

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. 

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. 

Molecules with structures as diverse as carbamoyl-phosphate, tryptophan, and cytidine triphosphate are feedback inhibitors of the E. coli glutamine synthase. The feedback inhibition is cumulative, with each metabolite exerting a partial inhibition on the enzyme. Why would complete inhibition of the glutamine synthase by a single metabolite be metaboli-cally unsound ... 

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. 

Dihydroorotate dehydrogenase, the enzyme catalyzing the dehydrogenation of dihydroorotate to orotate (reaction 4 of the pathway Fig. 15-15), is located on the outer side of the inner mitochondrial membrane. This enzyme has FAD as a prosthetic group and in mammals electrons are passed to ubiquinone. The de novo pyrimidine pathway is thus compartmentalized dihydroorotate synthesized by trifunctional DHO synthetase in the cytosol must pass across the outer mitochondrial membrane to be oxidized to orotate, which in turn passes back to the cytosol to be a substrate for bifunctional UMP synthase. Mammalian cells contain two carbamoyl phosphate synthetases the glutamine-dependent enzyme (CPSase II) which is part of CAD, and an ammonia-dependent enzyme (CPSase /) which is found in the mitochondrial matrix, and which is used for urea and arginine biosynthesis. Under certain conditions (e.g., hyperammonemia), carbamoyl phosphate synthesized in the matrix by CPSase I may enter pyrimidine biosynthesis in the cytosol. 

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). 

The urea cycle enzymes are controlled in the short term by the concentrations of their substrates. Carbamoyl phosphate synthetase I is also allosterically activated by N-acetylglutamate. This latter molecule is a sensitive indicator of the cell s glutamate concentration. (Recall that a significant amount of NH4 is derived from glutamate.) N-acetylglutamate is produced from glutamate and acetyl-CoA in a reaction catalyzed by N-acetylglutamate synthase. 

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