Citrulline and ornithine

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. 

Several standard and nonstandard amino acids act as metabolic intermediates. For example, arginine, citrulline, and ornithine (Figure 5.6) are components of the urea cycle (Chapter 15). The synthesis of urea, a molecule formed in vertebrate livers, is the principal mechanism for the disposal of nitrogenous waste. 

The first two reactions in the biochemical pathway that converts NFL,+ to urea (i.e., the formation of carbamoyl phosphate and citrulline) occur in the mitochondrial matrix. Subsequent reactions that convert citrulline to ornithine and urea occur in the cytosol. Both citrulline and ornithine are transported across the inner membrane by specific carriers. 

Many amino acids, such as citrulline and ornithine (which are found in the urea cycle), are not building blocks of proteins. Other nonstandard amino acids, such as hydroxyproline, are formed after translation by posttranslational modification. When discussing amino acids and translation, the magic number was always 20. Only 20 standard amino acids were put onto tRNA molecules for protein synthesis. In the late 1980s, another amino acid was found in proteins from eukaryotes and prokaryotes alike, including humans. It is selenocysteine, a cysteine residue in which the sulfur atom has been replaced by a selenium atom. 

Recall Describe citrulline and ornithine based on their similarity to one of the 20 standard amino acids. 

The resistant membranes in the CMC originate particularly from intracellular-membrane-associated regions, which form an envelope lining of cortical and cuticle cells [16]. An important difference between resistant membranes in cuticle and cortical cells is the presence of citrulline and ornithine in the cuticle membranes [166]. Some of the common features in the... 

Putrescine, citrulline, and ornithine are products of arginine metabolism. The molecular formulas are given for the form in which each exists at pH = 7. The net charge corresponding to each formula is zero, +1, and +2. Suggest a reasonable structure for each species. 

Some proteins may contain derivatives of common amino acids, e.g. collagen contains 5-hydroxy-lysine (Figure 1.6). Some amino acids which do not occur in proteins may function in cellular metabolism, e.g. citrulline and ornithine (Section 16.5). D-Amino acids occur alongside L-amino acids in some peptide antibiotics, e.g. valinomycin and actinomycin D, and in the peptidoglycan of the bacterial cell wall (Figure 3.10b). 

The enzymes of the urea cycle, however, are not only expressed in the liver, but also in other tissues and cell types. In fact, it is believed that the urea cycle evolved from the arginine metabolic pathway present in lower organisms (Takiguchi et al, 1989). This difference in function between urea and arginine synthesis is reflected by the different tissue localization, function and regulation of the enzymes of the urea cycle and other enzymes involved in the metabolism of the urea cycle intermediates, arginine, citrulline and ornithine. 

S-Citrulline (2>amino-5-ureidopentanoic acid) [372-75-8] M 175.2, m 222°, [o] +24.2° (in 5M HCl), pK 9.71. Likely impurities are arginine, and ornithine. Crystd from water by adding 5 volumes of EtOH. Also crystd from water by addn of MeOH. 

L-Ornithine transcarbamoylase catalyzes transfer of the carbamoyl group of carbamoyl phosphate to ornithine, forming citrulline and orthophosphate (reaction 2, Figure 29-9). While the reaction occurs in the mitochondrial matrix, both the formation of ornithine and the subsequent metabolism of citmlline take place in the cytosol. Entry of ornithine into mitochondria... 

The interaction with both synthetic and naturally occurring amino acids has been studied extensively glycine (138, 173, 219-221), a-(173, 219) and /3-alanine (138, 220), sarcosine (219), serine (222), aspartic acid (138, 173, 222-226), asparagine (222), threonine (222), proline (219), hydroxyproline (219), glutamic acid (138, 222-225), glutamine (222), valine (219, 227), norvaline (219), methionine (222, 226), histidine (228, 229), isoleucine (219), leucine (219, 230), norleu-cine (219), lysine (222), arginine (222), histidine methyl ester (228), phenylalanine (138, 222), tyrosine (222), 2-amino-3-(3,4-dihydroxy-phenyl jpropanoic acid (DOPA) (222), tryptophan (222), aminoiso-butyric acid (219), 2-aminobutyric acid (219,231), citrulline (222), and ornithine (222). 

Cohen and Grisolia then concentrated on the first step in the reaction, obtaining citrulline from ornithine. The reaction appeared to depend on oxygen, a requirement traced to the need for high concentrations of ATP. Physiologically the formation of urea occurs at very low levels of ammonia, which is extremely toxic as it is also lipid soluble and enters cells very easily. Cells are not very effectively buffered against OH. ... 

Citrulline enters the cytoplasm, and ornithine returns to the mitochondria. 

The concept of the ornithine cycle arose from the observation that ornithine, citrulline and arginine stimulated urea production in the presence of ammonia without themselves being consumed in the process. 

This enzyme [EC 3.5.3.12], also known as agmatine imi-nohydrolase, catalyzes the hydrolysis of agmatine to produce A/ -carbamoylputrescine and ammonia. The plant enzyme also catalyzes the reactions of EC 2.1.3.3 (ornithine carbamoyltransferase), EC 2.1.3.6 (putrescine car-bamoyltransferase) and EC 2.122 (carbamate kinase), thereby functioning as a putrescine synthase, converting agmatine and ornithine into putrescine and citrulline, respectively. 

This enzyme [EC 2.1.3.3], also known as ornithine car-bamoyltransferase and citrulline phosphorylase, catalyzes the reaction of carbamoyl phosphate with L-orni-thine to produce L-citrulline and orthophosphate. 

Major interfering substances found in the crude toxin preparation are citrulline, ethanolamine, ornithine, lysine and arginine. 

Canaline is a potent inhibitor of all seven pyridoxal phosphate-containing enzymes studied by Rahiala et (27) but it lacks adverse effects on three ornithine-utiTTzing enzymes lacking a Bg cofactor. Finally, in jack bean, Canavalia ensiformis, ornithine carbamoyl transferase can form 0-ureido-L-homoserine from canaline and carbamoyl phosphate as it does citrulline from ornithine and carbamoyl phosphate. Nevertheless, neither compound inhibited formation of the reaction products (31). 

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. 

About half of the NPN of milk is accounted for by urea. Orotic acid is a particular hallmark of the milks of ruminants milks of other species contain little if any of it (Larson and Hegarty 1977). The free amino acids constituting the a-amino N fraction in Table 1.6 include those that are also found in proteins, as well as ornithine, citrulline, and cx-amino butyric acid. Quantitative analyses of the mixture of free amino acids have been published (Deutsch and Samuelsson 1958 Armstrong and Yates 1963 Rassin et al. 1978). 

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. 

Grisolia and Towne (20) first proposed that an intermediate was formed prior to the synthesis of carbamyl-P. The idea of an intermediate was further strengthened by later evidence (35) regarding the increase in synthetic activity that follows the preincubation of the rat liver enzyme with acetyl glutamate, ATP, and Mg+2, and by the demonstration that, after preincubation of the enzyme with acetyl glutamate, ATP, Mg+2, and HC03", followed by the removal of ATP from the incubation mixtures, some citrulline was still synthesized upon addition of ornithine and ornithine transcarbamylase. 

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