Citrulline, urea cycle

See also Table 5.1, Genetic Code, Asparagine, Citrulline, Urea Cycle, Transamination in Amino Acid Metabolism, Citric Acid Cycle Intermediates in Amino Acid Metabolism, Essential Amino Acids... 

M.p. 222 C. Soluble in water, insoluble in alcohol. Citrulline is an intermediate in the urea cycle in the excretion of excess nitrogen from the body. 

All defects in urea synthesis result in ammonia intoxication. Intoxication is more severe when the metabolic block occurs at reactions 1 or 2 since some covalent linking of ammonia to carbon has already occurred if citrulline can be synthesized. Clinical symptoms common to all urea cycle disorders include vomiting, avoidance of high-protein foods, intermittent ataxia, irritability, lethargy, and mental retardation. The clinical features and treatment of all five disorders discussed below are similar. Significant improvement and minimization of brain damage accompany a low-protein diet ingested as frequent small meals to avoid sudden increases in blood ammonia levels. 

In 1930 Wada isolated citrulline from watermelon. It was an obvious candidate as an intermediate in the urea cycle. When tested by Krebs it was catalytic only at high concentrations. Citrulline is not normally detectable in liver. 

Arginine is converted to citrulline, which is released into the blood. This protects arginine from uptake and degradation in the urea cycle in the liver. Citrulline is released into the blood and transported to the kidney, where it is converted back to arginine (Chapter 8). 

Only a few important representatives of the non-proteinogenic amino acids are mentioned here. The basic amino acid ornithine is an analogue of lysine with a shortened side chain. Transfer of a carbamoyl residue to ornithine yields citrulline. Both of these amino acids are intermediates in the urea cycle (see p.l82). Dopa (an acronym of 3,4-dihydroxy-phenylalanine) is synthesized by hydroxyla-tion of tyrosine. It is an intermediate in the biosynthesis of catecholamines (see p.352) and of melanin. It is in clinical use in the treatment of Parkinson s disease. Selenocys-teine, a cysteine analogue, occurs as a component of a few proteins—e.g., in the enzyme glutathione peroxidase (see p.284). 

Citrulline is transported out of the mitochondria to the cytosol, where the other three reactions of the urea cycle take place. 

Fig. 6.14. Biosynthesis of NO. The starting point of NO synthesis is arginine. Arginine is converted by NO synthase, together with O, and NAD PH, to NO and citruUine. Arginine can be regenerated from citrulline via reactions of the urea cycle.

A series of supernatant transfer studies uncovered the fact that HC also produce NO, and in relatively large quantities (Curran et al., 1989). A supernatant generated from KC 8 hr after exposure to LPS and interferon-gamma (IFN-y) stimulated HC -NO biosynthesis and caused a simultaneous decrease in protein synthesis. As seen in the coculture model, a 6- to 8-hr delay in measurable NOj + N03 synthesis was seen in the HC exposed to conditioned KC supernatant. However, unlike KC or KC + HC where equimolar concentrations of citrulline were released into the culture supernatant, no increase in citrulline release paralleled the N02 + N03 synthesis by stimulated HC. It is most likely that the citrulline enters the HC urea cycle and is not secreted. 

FIGURE 3-8 Uncommon amino acids, (a) Some uncommon amino acids found in proteins. All are derived from common amino acids. Extra functional groups added by modification reactions are shown in red. Desmosine is formed from four Lys residues (the four carbon backbones are shaded in yellow). Note the use of either numbers or Creek letters to identify the carbon atoms in these structures, (b) Ornithine and citrulline, which are not found in proteins, are intermediates in the biosynthesis of arginine and in the urea cycle. 

The carbamoyl phosphate, which functions as an activated carbamoyl group donor, now enters the urea cycle. The cycle has four enzymatic steps. First, carbamoyl phosphate donates its carbamoyl group to ornithine to form citrulline, with the release of Pj (Fig. 18-10, step ). Ornithine plays a role resembling that of oxaloacetate in the citric acid cycle, accepting material at each turn of the cycle. The reaction is catalyzed by ornithine transcarbamoylase, and the citrulline passes from the mitochondrion to the cytosol. 

As we noted in Chapter 16, the enzymes of many metabolic pathways are clustered (p. 605), with the product of one enzyme reaction being channeled directly to the next enzyme in the pathway. In the urea cycle, the mitochondrial and cytosolic enzymes appear to be clustered in this way. The citrulline transported out of the mitochondrion is not diluted into the general pool of metabolites in the cytosol but is passed directly to the active site of argininosuccinate synthetase. This channeling between enzymes continues for argininosuccinate, arginine, and ornithine. Only urea is released into the general cytosolic pool of metabolites. 

Ammonia is highly toxic to animal tissues. In the urea cycle, ornithine combines with ammonia, in the form of carbamoyl phosphate, to form citrulline. A second amino group is transferred to citrulline from aspartate to form arginine—the immediate precursor of urea. Arginase catalyzes hydrolysis of arginine to urea and ornithine thus ornithine is regenerated in each turn of the cycle. 

Formation of citrulline Ornithine and citrulline are basic amino acids that participate in the urea cycle. [Note They are not incorporated into cellular proteins, because there are no codons for these amino acids (see p. 429).] Ornithine is regenerated with each turn of the urea cycle, much in the same way that oxaloacetate is regenerated by the reactions of the citric acid cycle (see p 109). The release of the high-energy phosphateof carbamoyl phosphate as inorganic phosphate drives the reaction in the forward direction. The reaction product, citrulline, is trans ported to the cytosol. 

Correct answer = D. Methionine is the precursor of cysteine. An increase in gluconeogenesis releases increased ammonia and results in increased urea production. The essential amino acids leucine and lysine are ketogenic. Ornithine and citrulline are amino acids that are intermediates in the urea cycle, but are not found in tissue proteins. 

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. 

Hyperammonemia has occurred during parenteral nutrition as a component of therapy for renal insufficiency (905). The hyperammonemia presented as a change in mental status, developing about 3 weeks after initiation of parenteral nutrition therapy in most cases the episodes are of increasing duration and paroxysmal. In three of the patients, serum amino acid analysis in the acute phase showed reduced concentrations of ornithine and citrulline (the respective substrate and product of condensation with carbamyl phosphate at its entry into the urea cycle). Concentrations of arginine, the precursor to ornithine, were raised. 

In the urea cycle ammonia is first combined with C02 to form carbamoyl phosphate. This then combines with ornithine to form citrulline. Citrulline then condenses with aspartate, the source of the second nitrogen atom in urea, to form argininosuccinate. This compound is in turn split to arginine and fumarate, and the arginine then splits to form urea and regenerate ornithine The first two reactions take place in the mitochondria of liver cells, the remaining three in the cytosol. 

The entry of activated ammonia into the urea cycle occurs by the ornithine transcarbamoylase reaction where the carbamoyl group is transferred to the side chain amino group of the non-protein amino acid, ornithine. Ornithine has five carbons its carbon chain therefore has the same length as that of arginine. The product of the ornithine transcarbamoylase reaction is the amino acid citrulline. 

Enzyme inhibition can be reversed by supplementation of arginine or citrulline.34 106 Pathovars of P. syringae were shown to inhibit Escherichia coli growth, an effect reversed by L-arginine, but not by L-citrulline or L-glutamine.121 This suggested that the site of action of the toxin produced is involved in the conversion of citrulline to arginine in the urea cycle. 

The most abundant amino add in the human organism does not occur in proteins and does not have a carboxyl group. Its addic residue is the sulfonate group, and its name is taurine (N+H3-CH2-CH2-S03 ). It occurs in the free state (exact function often unknown) and in bile salts, in which it plays an important role in fat digestion and absorption (see Chapters 9 and 19). Other amino acids that do not occur in proteins are ornithine and citrulline. They are important intermediates in the urea cycle described in Chapter 20. 

A number of amino acid transport disorders may be associated with one or several of the systems described in Table 20.4. These are characterized by the excretion of amino acids in the urine but no increase in amino acid levels in the bloodstream. They are usually of hereditary origin. The most common disorder is cystinuria, characterized by the excretion of cystine. Because cystine is only slightly water soluble, cystinuria is often accompanied by the deposition of cystine-containing stones in the genitourinary tract. Cystinuria is apparently caused by a defect in the cationic amino acid transport system. Another disease that affects this system is lysinuric protein intolerance, which is associated with a failure to transport lysine, ornithine, arginine, and citrulline across membranes. Citrulline and ornithine are urea cycle intermediates (see later), and a disruption of their interorgan traffic results in hyperammonemia. 

It has been stated that the liver is the principal urea producer, yet several urea cycle intermediates are produced by other organs as well and must be moved to other organs to be processed. Thus, intestinal mucosal cells are able to convert ornithine to citrulline, but citrulline cannot be converted to arginine in that location. Citrulline must be transported to either the kidney or liver, where conversions to arginine and urea are possible. Kidney, on the other hand, cannot convert ornithine to citrulline. 

OTC deficiency is the most common urea cycle defect.As it is X linked, affected boys typically have severe disease with neonatal presentation as described in this chapter. The disease in women who carry an OTC mutation on one X chromosome ranges from severe early-onset disease to complete absence of symptoms. Furthermore, affected women may decompensate in the context of a metabolic stress such as an infection or following parturition. OTC-deficient patients have low plasma citrulline and high urine orotic acid. Confirmation of the diagnosis requires mutation analysis or a liver biopsy for enzymology. The carrier status of women is most accurately determined by mutation analysis. 

Citrulline is exchanged for ornithine across the inner mitochondrial membrane by ORNT-1. Ornithine is produced in the cytosol as the final step in the urea cycle and must be returned to the mitochondrial matrix for transcarbamoyla-tion by OTC. A second ornithine-citrulline antiporter (ORNT-2) is also expressed in the liver mitochondria and may attenuate the severity of disease in patients with HHH (Hyperammonemia, Hyperornithinemia, Homocitrullinuria) disease due to ORNT-1 deficiency. This disorder typically manifests later in life with intermittent hyperammonemic encephalopathy and protein aversion. Intramitochondrial ornithine deficiency causes both hyperammonemia and hyperornithinemia due to a lack of substrate for OTC. Homocitrullinuria occurs due to the use of lysine by OTC as an alternate substrate. The diagnosis is confirmed by mutation analysis. 

The final step of the urea cycle is the cleavage of arginine to release urea and regenerate ornithine. Ornithine then reenters the mitochondria via the ORNT-1 ornithine-citrulline antiporter. ARG-1 is a cytosolic homotrimeric enzyme of 35-kd monomers that is expressed in fiver and red blood cells. A second mitochondrial arginase (ARG-2) most likely plays a role in nitric oxide synthesis and is most abundant in brain, kidney, and prostate. ARG-1 deficiency is unique among the urea cycle deficiencies as patients do not present with hyperammonemia and encephalopathy but rather develop progressive spasticity of the lower limbs. Biochem-... 

Citrulline takes its name from the watermelon genus (Citrullus) in which it was first found in 1930. It was also discovered the same year as a bacterial degradation product of arginine. Krebs, who elucidated the form of the urea cycle, demonstrated that citrulline was the intermediate between ornithine and arginine. The urea cycle was the first metabolic cycle to be discovered. In Krebs words, it revealed a new pattern of the organization of metabolic processes. ... 

Ornithine and citrulline are amino acids, but they are not used as building blocks of proteins. The formation of NH4 + by glutamate dehydrogenase, its incorporation into carbamoyl phosphate, and the subsequent synthesis of citrulline take place in the mitochondrial matrix. In contrast, the next three reactions of the urea cycle, which lead to the formation of urea, take place in the cytosol. 

What about the other enzymes in the urea cycle Ornithine transcarbamoylase is homologous to aspartate transcarbamoylase and the structures of their catalytic subunits are quite similar (Figure 23.18). Thus, two consecutive steps in the pyrimidine biosynthetic pathway were adapted for urea synthesis. The next step in the urea cycle is the addition of aspartate to citrulline to form argininosuccinate, and the subsequent step is the removal of fumarate. These two steps together accomplish the net addition of an amino group to citrulline to form arginine. Remarkably, these steps are analogous to two consecutive steps in the purine biosynthetic pathway (Section 25.2 3). 

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