Carbamyl aspartic acid

Pyrimidine biosynthesis commences with a reaction between carbamyl phosphate and aspartic acid to give carbamyl aspartic acid which then nndergoes ring closure and oxidation to orotic acid. A reaction then occurs between orotic acid and 5-phosphoribosyl pyrophosphate to give orotidine-5-phosphate which on decarboxylation yields uridine-5-phosphate (UMP). By means of two successive reactions with ATP, UMP can then be converted into UTP and this by reaction with ammonia can give rise to cytidine triphosphate, CTP (11.126). 

The operation of the feedback mechanism in the cell can be described in the following manner if nucleic acid syntheris were proceeding at a rapid rate, the concentration of nucleotides in the cell, e.g. CMP, would decrease, relieve aspartate-carbamyl transferase (also called carbamyl-aspartic acid synthetase) reaction of any inhibition, and thus permit an increased rate of pyrimidine biosyntheris. If nucleic acid syntheris were slowed or halted, the CMP concentration would rise, inhibit the enzyme, and thereby decelerate pyrimidine biosynthesis. 

Figure 2 shows the main stages of pyrimidine nucleotides biosynthesis. In stage 1 aspartic add and carbamyl phosplmte (formed from ammonia, CO3 and ATP) condense to form carbamyl aspartic acid. This derivative cydizes with loss of water to form dihydroorotic add which is converted by a dehydrogenase to orotic add. The nudeotide of orotic acid (orotidylic add (OI )) is dien formed in... 

The brain enzyme has been purified over 1000-fold and shown to be homogeneous by ultracentrifugation and electrophoresis criteria (36) the activity ratio for acetyl-P over carbamyl-P remains unchanged with purification. This enzyme is one of the smallest on record the molecular weight from physical data is 13,200 and from amino acid analysis is 12,600 the amino acid composition of the enzyme is given in Table I. The terminal amino acid is aspartic acid (25). Cystine has not been detected. 

Reactions 3 and 4 indicate that with aspartic acid, aspartic transcarbamylase, and carbamyl-P or acetyl-P, either carbamyl aspartate or acetyl aspartate can be formed. Carbamyl aspartate is the first intermediate in the formation of pyrimidines, and acetyl aspartate, of unknown function, is the amino acid derivative present in the largest concentration in brain of most species (43). 

Prepare the amount ofcolor reagent you need—3 ml to he added to each tube containing 2 ml of carbamyl aspartate standard or enzyme assay mixture after addition of perchloric acid. Add 3 ml to each assay and standard tube and mix thoroughly. Cap the tubes with marbles or parafilm, carver them with aluminum foil, and store in a dark place at room temperature until the next lab period (15-48 hr is satisfactory). 

Conduct the assays for Series A, B, and C, initiating each reaction with 100 /id 0.036 M carbamyl phosphate, terminating after 30 min of reaction with 1 ml of 2% perchloric acid, and developing the color by adding 3.0 ml of freshly prepared color reagent as described above. Include a series of carbamyl aspartate standards also as described above. 

Dihydroorotase catalyzes the intramolecular cyclization of 7V-carbamyl-L-aspartic acid to L-dihydroorotic acid. In mammals, the activity is present in a trifunctional enzyme that catalyzes the first three steps in the de novo synthesis of pyrimidine nucleotides. 

The first step in the formation of urea from ammonia is its combination with bicarbonate to form carbamyl phosphate (Fig. 1). This contributes only one nitrogen atom to urea, the other being donated by aspartic acid in the third step of the pathway. A -Acetylglutamate is required as cofactor, and the presence of Mg is essential, ATP being converted to ADP in the process. The reaction is catalyzed by carbamyl phosphate synthetase (carbamate kinase EC 2.7.2.2). It has been shown that there are probably two forms of this enzyme, at least in rat liver. One is ammonia dependent, is primarily associated with mitochondria, and may be the enzyme responsible for the formation of carbamyl phosphate in the synthesis of urea. The other, which is glutamine dependent, is probably mainly extramitochondrial and may supply the carbamyl phosphate used... 

The utilization of ammonia resulting from the combination of carbamyl phosphate with aspartic acid, the initial reaction for the synthesis of the pyrimidine nucleotides, continues only as long as there is a requirement for them (Fig. 3). Regulation of this biosynthetic pathway is probably by way of feedback inhibition of aspartate transcarbamylase. The rat liver enzyme is inhibited by uridine, cytidine or thymidine or such derivatives as CMP, UTP, or TMP, all intermediates or products of this pathway (B8). This is not the only enzyme of the pathway which may be subject to feedback regulation. Dihydroorotase from rat liver is also inhibited by some pyrimidines and purines (B9). 

ASA + NHa —> ornithine + carbamyl aspartate Carbamyl aspartate NH3 — guanidinosuccinic acid Guanidinosuccinic acid — urea + aspartate... 

One of the two —NH2 groups comes in from ammonia, which reacts with CO2 and ATP to form carbamyl phosphate, NH2.COO-phosphate, one of the feed compounds for the cycle. This then reacts with ornithine, shedding the phosphate and forming another new amino acid, citmlline. The second —NH2 comes in, not from ammonia but from aspartic acid (see Topic 23). Reaction of aspartic acid with cit-rulline and another ATP gives argininosuccinic acid (do not worry about either the... 

Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is an allosteric enzyme which controls the first step of p5oimidine de novo biosynthesis. It catalyzes the condensation of L-aspartic acid with carbamyl phosphate to produce carbamoyl-L-aspartate... 

The primary step in the urea cycle is the synthesis of carbamyl phosphate from ammonia and carbon dioxide (11.76). This first stage, and the later stage of synthesis of arginosuccinic acid from citrulline and aspartic acid, both require the transfer of energy from ATP hydrolysis. The pyrophosphate formed in the latter reaction is itself hydrolysed which, together with the former reaction. 

Historically, carbamyl aspartate was recognized as a likely intermediate in pyrimidine biosynthesis because (a) this compound is an assembly of two elementary precursors of the pyrimidine ring, (b) carbamyl aspartate would satisfy the nutritional requirement of L. bvlgaricus 09 for orotate, and (c) labeled carbamyl aspartate was incorporated into ribonucleic acid pyrimidines in L. bulgaricus and, as well, served as an orotate precursor in liver slice trapping experiments such as those mentioned above. 

The steps in the conversion of carbamyl aspartate to orotate became apparent in work by Lieberman and Kornberg (8), who studied the reverse process, the degradation of orotic acid by an orotate-fermenting bacterium, Zymobacterium oroticum. Intact cells of this organism degraded orotate to NHa, CO2, acetic acid, and dicarboxylic acids however, in broken cell preparations, the degradation of orotate did not proceed to that extent and two intermediates were isolated, dihydroorotate and carbamyl aspartate ... 

L-Arginine originates from L-ornithine, carbamyl phosphate and the amino group of L-aspartic acid (Fig. 236). 

As early as 1949, it was demonstrated that injected or " C-labeled orotic acid was readily incorporated into DNA and RNA of mammalian tissue, indicating that orotic acid is a precursor of nucleic acid pyrimidine. The next step in pyrimidine biosynthesis is the formation of the first nucleotide in the sequence. It involves the reaction between ribosyl pyrophosphate and orotic acid to yield 5 -orotidylic acid the reaction is catalyzed by orotidylic pyrophosphorylase. Thus, the first steps of pyrimidine biosynthesis differ from the early steps of purine biosynthesis in at least two ways. Orotic acid, instead of being synthesized atom by atom as is the case for the purine ring, is made from the condensation of rather large molecules, namely, carbamyl phosphate and aspartic acid. Furthermore, all the steps of purine biosynthesis occur at the level of the nucleotide, but the the pyrimidine ring is closed at the level of the base. 

The first step in pyrimidine biosynthesis is the reaction catalyzed by aspartic transcarbamylase, a reaction in which the carbamyl group of carbamyl phosphate is transferred to aspartic acid to yield ureidosuccinic acid. In E. coli, the end products of the pyrimidine pathways, UTP and especially CTP, inhibit the transcarbamylase. Pardee and his associate, who discovered this important event, established that the site of action in the transcarbamylase molecule of the inhibitor is different from the site of action of the substrate. 

An early form of therapy involves eliminating the substrate either by excluding the substrate from the diet, as in phenylketonuria, or by administering drugs—such as penicillamine in Wilson s disease or allopurinol in gout. Orotic aciduria can be corrected by the administration of uridine, which provides the substrate for the biosynthesis of the nucleosides used in RNA and DNA synthesis and is also a substrate for the biosynthesis of inhibitors of the carbamyl aspartate synthetase, the first enzyme in the formation of orotic acid. By this feedback inhibition, the levels of orotic acid in the urine are reduced by the administration of uridine. 

Two separation procedures were used to identify all products that could be formed from [4-aspartate. With the HVPE the end-product, OA, could be separated from the substrate and all other products, including citric acid cycle intermediates. Malate and fuma-rate had the same mobility as carbamyl aspartate and DHO, respectively, but could be separated by TLC. The identity of OA was confirmed by conversion of eluted radioactivity with partially purified yeast OPRT and ODC to OMP and UMP. With brain cortex the rate of OA synthesis from aspartate was 52 12 and with liver 179 35 nmol/h per g wet tissue (means SD of 7 and 4 experiments, respectively) expressed per mg protein these values were 0.81 0.21 and 1.12 0.46, respectively. With both tissues about 10% of the label was found in citric acid cycle intermediates, and with cortex and liver about 1% and 10% of radioactivity was recovered as 002 ... 

Most of the ammonia (NH,) produced through deamination—removal of amino groups (NH,) from amino acids is converted to urea in the liver for excretion by the kidneys. To facilitate elimination, 1 mole of ammonia (NH,) combines with 1 mole of carten dioxide (CO,), another metatelic waste product. This compound is then phosphorylated to produce carbamyl phosphate. Carbamyl phosphate then combines with ornithine to form citrulline an intermediate in the urea cycle. The amino acid, aspartic acid, contributes another amino group (NH,), and citrulline is then converted to the amino acid arginine. Urea splits off from arginine forming ornithine, and the cycle is completed. Fig. U illustrates the urea cycle. 

The formation of carbamylaspartic acid from carbamyl phosphate and aspartic acid (Fig. 20) has been demonstrated in pigeon liver, several tissues of the rat, E. colt, and yeast (363S67). It appears that only one enz3rme was involved in thb interesting reaction 368) and it has been named ureidosuccinic (carbamylaspartic) acid synthetase 355). The reaction was essentially irreversible, even though slight reversal was shown with labeled substrates 357). 

The location of the metabolic block appears to be advantageous to cellular economy, sparing energy, and metabolites. The formation of carbamylaspartic acid was essentially an irreversible reaction and inhibition of the sequence at a subsequent step would be considered inefficient since carbamylaspartic acid would be produced whether it were needed or not. It is of further interest that the steps preceding the blocking point, the formation of aspartic acid and carbamyl phosphate, were freely reversible reactions. 

The Biosynthesis of the Pyrimidine Ring begins with aspartic acid and carbamyl phosphate. The latter is an energy-rich compound which reacts with the former to give carbamylaspartic acid. Ring closure consumes ATP and is in principle an acid amide formation (peptide synthesis). The intermediate dihydro-orotic acid is dehydrogenated to orotic acid, probably by action of a flavoprotein. Orotic acid is the key precursor of pyrimidine nucleotides. It reacts with phosphoribosyl pyrophosphate. The removal of pyrophosphate yields the nucleotide of orotic acid, whose enzymic decarboxylation produces uridine 5 -phosphate. Phosphorylation with ATP yields uridine pyrophosphate and, finally, uridine triphosphate. Beside the above pathway, there is the further possibility of converting free uracil and ribose 1-phosphate to the nucleoside and from there with ATP to the nucleotide. 

In other cases we encounter not induction but repression of enzyme synthesis by a metabolite in whose synthesis the enzyme is concerned (the same may also be inhibited in its activity by other components of the reaction sequence in which it it involved, see p. 252). The enzyme whose activity is lost or markedly reduced is not necessarily that which completes the synthesis of the repressor molecule (i.e. not that catalysing the last reaction of the biosynthetic pathway). For instance, uracil can suppress, in certain strains of the bacterium, Escherichia coU, the activity of the enzyme aspartate carbamyl-transferase which promotes the interaction between aspartic acid and carbamyl phosphate, a reaction which is the first step in the reaction sequence involved in pyrimidine biosynthesis. Further, the experimental evidence indicates that the uracil acts as a repressor by preventing synthesis of the enzyme. 

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