The Urea Cycle

In the oxidative deamination of glntamate, is the hydride ion transferred to the Re face or the Si face of NAD (Review Section 5.11.) 

The ammonia resulting from amino acid deamination is eliminated in one of three ways depending on the organism. Fish and other aquatic animals simply excrete the ammonia to their aqueous surroundings, but terrestrial organisms must first convert the ammonia into a nontoxic substance—either nrea for mammals or uric acid for birds and reptiles. 

The conversion of ammonia into nrea begins with its reaction with bicarbonate ion and ATP to give carbamoyl phosphate. The reaction is catalyzed by carbamoyl phosphate synthetase I and occurs by initial activation of HC03 by ATP to give carhoxy phosphate, followed by nucleophilic acyl substitution with ammonia to prodnce carbamate plus phosphate ion (Pj) as the leaving gronp. Subseqnent phosphorylation of carbamate by a second 

FIGURE 20.4 Mechanism of the formation of carbamoyl phosphate from bicarbonate. Bicarbonate ion is first activated by phosphorylation with ATP, and a nucleophilic acyl substitution with ammonia then occurs. 

Carbamoyl phosphate next enters the four-step urea cycle, whose overall result can be summarized as 

The urea cycle converts ammonia to urea, a nontoxic substance. 

One of the nitrogen atoms for urea synthesis comes from ammonia and the other is donated by aspartate. 

Urea formed in the liver is highly water-soluble and is carried by the blood to the kidneys where it is filtered and excreted in the urine. 

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

Flux of ammonia through the urea cycle is regulated by two factors  

However Henseleit showed there was a correlation between the concentration of ornithine and the magnitude of the effect on urea production. That the absolute amounts of ornithine were so small provided Krebs with arguments for the idea that the ornithine acted catalytically. 

The paper by Krebs and Henseleit (1932), Experiments on the Formation of Urea in Animal Bodies (Klinische Wochenschrift 11,759), contained the phrases... in the synthesis of urea in the living cell, ornithine acts like a catalyst. We therefore draw the conclusion. .. that the primary reaction for the synthesis of urea from ammonia is 

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. 

It was not until 1933 after Krebs had decided to leave Freiburg that he formulated urea synthesis in its now common cycle form  

This first appeared in Hildegard Mandershied s M.D. thesis, On the Formation of Urea in Vertebrates. (Holmes, 1991). 

Excess nitrogen is excreted as ammonia. Ammonotelic organisms excrete ammonia directly, uricotelic organisms excrete it as uric acid, and ureotelic organisms excrete it as urea. 

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 fumarate produced in the urea cycle can enter directly into the citric acid cycle and be converted into oxaloacetate. Oxaloacetate can then be either transaminated to aspartate which feeds back into the urea cycle, or be converted into citrate, pyruvate or glucose. 

Hyperammonemia is an increase in the levels of ammonia in the blood caused by a defect in an enzyme of the urea cycle. The excess ammonia is channeled into glutamate and glutamine with a deleterious effect on brain function. 

The urea cycle intermediate arginine can be condensed with glycine to form guanidinoacetate, which in turn is methylated by the methyl donor S-adenosyl methionine to creatine. The creatine is then phosphorylated to form creatine phosphate, a high-energy store found in muscle. 

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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. 

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. 

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... 

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

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. 

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... 

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). 

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. 

The first suggestion that substrates in carbohydrate oxidation might exert catalytic effects on the oxidation of other intermediates (cf.earlier demonstration of such action in the urea cycle by Krebs and Henseleit, 1932 see Chapter 6) arose from the work of Szent-Gyorgi (1936). He demonstrated that succinate and its 4C oxidation products catalytically stimulated the rate of respiration by muscle tissues. He also observed that reactions between the 4C intermediates were reversible and that if muscle was incubated with oxaloacetate, fumarate and malate made up 50-75% of the products, 2-oxoglutarate 10-25% and, significantly, 1-2% of the C was converted to citrate. These observations were... 

In the next 2 to 3 years further experiments, particularly by Eggleston, who had joined Krebs in January 1936, confirmed and extended the observations. Careful quantitative evaluation of the data indicated that citrate like fumarate (Szent-Gyorgi) and like ornithine in the urea cycle exerted a catalytic effect on muscle metabolism. If arsenite, which blocks 2-oxoglutarate oxidation, was added with citrate to a respiring pigeon-muscle preparation, 2-oxoglutarate accumulated. 

Lohman discovered ATP in muscles. Krebs and Henseleit. The urea cycle. Svedberg began studies with the ultracentrifuge. 

The ammonia liberated by GLDH does not itself enter the urea cycle it must first be combined with carbon dioxide to form carbamoyl phosphate. This is an energy (ATP) consuming reaction ... 

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

The reaction shown in Figure 8.6 is also important in the liver where glutamate dehydrogenase is involved in the catabolism of amino acids and the entry of nitrogen into the urea cycle, as explained in Chapter 6. 

In liver, aminotransferases ALT and AST can move the amino group from alanine arriving from muscle into aspartate, a direct donor of nitrogen into the urea cycle. 

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