Urea cycle

Humans expel most of their waste nitrogen as urea and their phosphorus as HjPOl , both of which are found in faeces and urine. 

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. 

In plants and some bacterial species, ammonia is ranoved as glutamate, with NADPH and glutamine dehydrogenase. 

Urea is synthesized from ammonia, carbon dioxide, and aspartate nitrogen using Kreb s Urea Cycle  

Carbamoyl Phosphate Synthetase I (CPS I) provides the substrate, carbamoyl phosphate, for the urea cycle. 

A total of four ATP equivalents has been consumed. How many ATP equivalents are then required to convert the nitrogen of two amino acids into urea The flow diagram supplied below may help in this calculation  

To solve this problem we can first note the four ATP equivalents consumed in the biosynthesis of one urea from two amino acids. But the fumerate produced must be taken back to regenerate the oxalacetate used to pick up the nitrogen from one amino acid)  

This will provide an NADH via malate DH. This is equivalent to 2.5 ATP s, so we have -4 + 2.5 = -1.5. Next, while the second nitrogen enters via two transaminations through aspartate  

Amino acids are mainly broken down in the liver. Ammonia is released either directly or indirectly in the process (see p. 178). The degradation of nucleobases also provides significant amounts of ammonia (see p. 186). 

Ammonia (NH3) is a relatively strong base, and at physiological pH values it is mainly present in the form of the ammonium ion NH4 (see p. 30). NH3 and NH4 are toxic, and at higher concentrations cause brain damage in particular. Ammonia therefore has to be effectively inactivated and excreted. This can be carried out in various ways. Aquatic animals can excrete NH4 directly. For example, fish excrete NH4 via the gills (ammonotelic animals). Terrestrial vertebrates, including humans, hardly excrete any NH3, and instead, most ammonia is converted into urea before excretion ureotelic animals). Birds and reptiles, by contrast, form uric acid, which is mainly excreted as a solid in order to save water uricotelic animals). 

The reasons for the neurotoxic effects of ammonia have not yet been explained. It may disturb the metabolism of glutamate and its precursor glutamine in the brain (see p. 356). 

Urea (H2N-CO-NH2) is the diamide of carbonic acid. In contrast to ammonia, it is neutral and therefore relatively non-toxic. The reason for the lack of basicity is the molecule s mesomeric characteristics. The free electron pairs of the two nitrogen atoms are delocalized over the whole structure, and are therefore no longer able to bind protons. As a small, uncharged molecule, urea is able to cross biological membranes easily. In addition, it is easily transported in the blood and excreted in the urine. 

Urea is produced only in the liver, in a cyclic sequence of reactions (the urea cycle) that starts in the mitochondria and continues in the cytoplasm. The two nitrogen atoms are derived from NH4 (the second has previously been incorporated into aspartate see below). The keto group comes from hydrogen carbonate (HC03 ), or CO2 that is in equilibrium with HC03.  

Function To provide a route to dispose of the amino groups from amino acids during their metabolism. 

Connections From amino groups of amino acids through glutamate and glutamate dehydrogenase 

From ammonia tWugh carbamoyl phosphate synthetase To urea 

Regulation Primarily by availability of amino groups and ammonia Equation  

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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. 

Urea occurs in the urine of all mammals and in small quantities in the blood of mammals and fish (see urea cycle). 

In 1937 Krebs found that citrate could be formed in muscle suspensions if oxaloacetate and either pyruvate or acetate were added. He saw that he now had a cycle, not a simple pathway, and that addition of any of the intermediates could generate all of the others. The existence of a cycle, together with the entry of pyruvate into the cycle in the synthesis of citrate, provided a clear explanation for the accelerating properties of succinate, fumarate, and malate. If all these intermediates led to oxaloacetate, which combined with pyruvate from glycolysis, they could stimulate the oxidation of many substances besides themselves. (Kreb s conceptual leap to a cycle was not his first. Together with medical student Kurt Henseleit, he had already elucidated the details of the urea cycle in 1932.) The complete tricarboxylic acid (Krebs) cycle, as it is now understood, is shown in Figure 20.4. 

One step in the urea cycle for ridding the body of ammonia is the conversion of argininosuccinate to the amino acid arginine plus fumarate. Propose a mechanism for the reaction, and show the structure of arginine. 

Watford, M. (1991). The urea cycle A two system compartment system. Essays Biochem. 26.49-48. 

If the effect of water stress is to alter regulation of the pathway such that the rate constant for reaction A G is increased or A CP is decreased (which would have an overall effect of conserving nitrogen), then the fractionation at G can be shown to be thereby increased. At present this is speculative, but in fact explanations for the water-stress effect using flow-models are rather constrained. For example, it is not possible to relate what might happen at the kidneys (e.g., resorption of urea) to the amino acid body pool, since the urea cycle is non-reversible. It should be possible to design experiments that test this suggestion. 

While ammonia, derived mainly from the a-amino nitrogen of amino acids, is highly toxic, tissues convert ammonia to the amide nitrogen of nontoxic glutamine. Subsequent deamination of glutamine in the liver releases ammonia, which is then converted to nontoxic urea. If liver function is compromised, as in cirrhosis or hepatitis, elevated blood ammonia levels generate clinical signs and symptoms. Rare metabolic disorders involve each of the five urea cycle enzymes. 

Urea biosynthesis occurs in four stages (1) transamination, (2) oxidative deamination of glutamate, (3) ammonia transport, and (4) reactions of the urea cycle (Figure 29-2). 

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... 

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... 

METABOLIC DISORDERS ARE ASSOCIATED WITH EACH REACTION OF THE UREA CYCLE... 

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. 

Gene therapy for rectification of defects in the enzymes of the urea cycle is an area of active investigation. Encouraging preliminary results have been obtained, for example, in animal models using an adenoviral vector to treat citrullinemia. 

Hepatic urea synthesis takes place in part in the mitochondrial matrix and in part in the cytosol. Inborn errors of metabolism are associated with each reaction of the urea cycle. 

Deficiency of a Urea Cycle Enzyme Results in Excretion of Pyrimidine Precursors... 

Nitrogen is dumped into the urea cycle by transamination to make Asp or Glu or by deamination to make ammonia. 

The nitrogen contained in the amino acids is usually disposed of through the urea cycle. One of the early, if not the first, steps in amino acid catabolism involves a transamination using oxaloacetate or a-ketoglutarate as the amino-group acceptor. This converts the amino acid into a 2-keto acid, which can then be metabolized further. 

The result is that the amino groups can be dumped out as alanine (the transamination product of pyruvate). In the liver and kidney, alanine is transaminated to yield pyruvate and glutamate. As in the Cord cycle, the pyruvate is converted to glucose by the liver and is shipped out. The glutamate is fed into the urea cycle-nitrogen disposal system to get rid of the excess nitrogen. 

The urea cycle is essential for the detoxification of ammonia 678 Urea cycle defects cause a variety of clinical syndromes, including a metabolic crisis in the newborn infant 679 Urea cycle defects sometimes result from the congenital absence of a transporter for an enzyme or amino acid involved in the urea cycle 680 Successful management of urea cycle defects involves a low-protein diet to minimize ammonia production as well as medications that enable the excretion of ammonia nitrogen in forms other than urea 680... 

Urea cycle defects Failure to convert ammonia to urea via urea cycle (Fig. 40-5). Coma, convulsions, vomiting, respiratoryfailure in neonate. Often mistaken for sepsis of the newborn. Mental retardation, failure to thrive, lethargy, ataxia and coma in the older child. Associated with hyperammonemia and abnormalities of blood aminogram Low protein diet Acylation therapy (sodium benzoate, sodium phenylacetate) Arginine therapy in selected syndromes Hepatic transplantation... 

Treatment of aminoacidurias with a low-protein diet may influence brain chemistry. It should be emphasized that the treatment of the patient with an aminoaciduria may affect brain chemistry, perhaps in an adverse manner. Nearly all patients receive a low-protein diet. Indeed, undiagnosed patients sometimes avoid consumption of protein, which they feel intuitively can cause lethargy, headache, nausea and mental confusion. As dietary protein declines, the intake of carbohydrate frequently increases. The concomitant rise of endogenous insulin secretion favors an increase in the ratio of the concentration of blood tryptophan to that of other amino acids, thereby promoting the entry of tryptophan to the brain. The latter amino acid is precursor to brain serotonin, which tends to increase. This physiology is known to be operative in patients with urea cycle defects. 

The urea cycle is essential for the detoxification of ammonia. The urea cycle (Fig. 40-5) converts ammonia to urea (10-20g/day in the healthy adult). A urea cycle enzymopathy, whether associated with cirrhosis or an inherited metabolic defect, often causes a hyperammone-mic encephalopathy and irreversible brain injury (see also Ch. 34). 

Urea cycle defects cause a variety of clinical syndromes, including a metabolic crisis in the newborn infant. 

Severe urea cycle defects become manifest in infants with a severe syndrome of coma, convulsions and vomiting during the first few days of life. Clinical confusion with septicemia is common, and many infants are treated futilely with antibiotics. Hyperammonemia is usually severe, even in excess of 1 mmol/1 (normal in term infants <100 xmol/l). 

Diagnosis of a urea cycle defect in the older child can be elusive. Patients may present with psychomotor retardation, growth failure, vomiting, behavioral abnormalities, perceptual difficulties, recurrent cerebellar ataxia and headache. It is therefore essential to monitor the blood ammonia in any patient with unexplained neurological symptoms, but hyperammonemia is inconstant with partial enzymatic defects. Measurement of blood amino acids and urinary orotic acid is indicated. 

Ornithine transcarbamylase deficiency. This is the most common of the urea cycle defects. Presentation is variable, ranging from a fulminant, fatal disorder of neonates to a schizophrenic-like illness in an otherwise healthy adult. Males characteristically fare more poorly than do females with this X-linked disorder because of random inactivation (lyonization) of the X chromosome. If inactivation affects primarily the X chromosome bearing the mutant OTC gene, then a more favorable outcome can be anticipated. Conversely, the unfavorably lyonized female has a more active disease. 

Urea cycle defects sometimes result from the congenital absence of a transporter for an enzyme or amino acid involved in the urea cycle. 

We begin this overview of manganese biochemistry with a brief account of its role in the detoxification of free radicals, before considering the function of a dinuclear Mn(II) active site in the important eukaryotic urea cycle enzyme arginase. We then pass in review a few microbial Mn-containing enzymes involved in intermediary metabolism, and conclude with the very exciting recent results on the structure and function of the catalytic manganese cluster involved in the photosynthetic oxidation of water. 

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