LECTURE 34: UREA CYCLE

Fate of Urea Urea diffuses from the liver and transported in the blood to the kidneys where it is filtered and excreted in the urine. A portion of urea diffuses from blood into intestine and cleaved to NH3 and CO2 by bacteria urease. This ammonia is partly lost in feces and is primarily reabsorbed into the blood.

[Note: In kidney failure, plasma urea levels are higher which lead to a greater transfer of urea from the blood into intestine. The urease activity becomes a clinically important source of ammonia and contributes to hyperammonemia. Oral administration of neomycin reduces the number of these bacteria].

TRANSAMINASES PARTICIPATE IN THE FLOW OF NITROGEN FROM AA TO AMMONIA AND UREA. -Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are the two most important aminotransferases.

-Ammonia is released from Glutamate by Oxidative Deamination.

4. In the fourth reaction of the urea cycle, arginosuccinase,also known as arginosuccinate lyase, catalyzes conversion of arginosuccinate to arginine and fumarate. The urea cycle is linked to the citric acid cycle through the production of fumarate. Amino acid catabolism is therefore coupled to energy production. 5. The last reaction of the urea cycle is catalyzed by the enzyme arginase, which is found only in liver cells. Arginase catalyzes hydrolysis of the guanidine group of arginine, releasing urea and ornithine. Ornithine then continues in the urea cycle.

Enzymes that catalyze the first four reactions of the urea cycle are found in the liver, kidney, and intestinal mucosa, but arginase is found only in the liver. The arginine found in the kidney and the intestinal mucosa is used in the biosynthesis of proteins.

1. The Overall Reaction Aspartate + NH3 + CO2 + 3ATP--> UREA + FUMARATE + 2ADP + 2Pi + AMP + 2Pi + 3 water

a. The synthesis of each molecule of urea requires 4 high energy phosphates from 3 molecules of ATP. Therefore, the synthesis of Urea is irreversible with large negative free energy. b. One nitrogen is donated from ammonia and one from aspartate, but both are derived from glutamate, as shown above. c. The carbon and oxygen atoms are derived from HCO3-, derived from the TCA cycle. d. The urea cycle is found exclusively in the liver, and thus the levels of arginase are high only in liver. In other tissues arginine is used for synthesis of proteins.This is true for all animals.

.Transamination: funneling of amino group to glutamate. -Transamination: The α-amino group from α-amino acid is transferred to α-ketoglutarate. The products are an α-keto acids (derived from the original amino acid) and glutamate. The reactions, which are readily reversible, use pyridoxal phosphate (PLP) as a cofactor. The enzymes are called transaminases or aminotransferases. -These enzymes are present in the cytoplasm and mitochondria of cells throughout the body especially those of liver, kidney, muscle and intestine.

-Two most important aminotransferases are alanine aminotransferase (ALT) and aspartate aminotransferase (AST). -All amino acids with the exception of lysine, proline and threonine participates in transamination at some point of their catabolism (these two amino acids loose their α-amino acid via deamination).

.Oxidative Deamination of Amino Acids. Glutamate is the only amino acid which uniquely undergoes oxidative deamination reaction catalyzed by Glutamate Dehydrogenase. In this reaction glutamate is converted to α-Ketoglutarate and liberates amino acid as free NH4+.

Direction of the reaction: The direction of the reaction depends upon the relative concentrations of glutamate, α-ketoglutarate, ammonia, and the ratio of the oxidized to reduced coenzymes.

Additional mechanism for Deamination of specific Amino Acids The primary route of amino group removal is via transamination, but there are additional enzymes capable of removing the α-amino group. Point: there are more deamination reactions. Do not have to know this reaction and he said it wasn't really imp.

(A) Amino acid oxidases such as L-amino acid oxidase produce ammonia and an a-keto acid directly, using flavin mononucleotide (FMN) as a cofactor. The reduced form of the flavin must be regenerated using molecular oxygen, and this reaction is one of several that produce H2O2. The peroxide is decomposed by catalase. (B) A second means of deamination is possible only for hydroxyamino acids (serine and threonine), through a dehydratase mechanism that involves a dehydration followed by the readdition of water and loss of the amino group as ammonia

TREATMENT OF HYPERAMMONEMIA. Because ammonia is poorly cleared by the kidneys, its removal from the body must be expedited by formation of compounds with high renal clearance.

-A low-protein, alcohol free diet is the mainstay of treatment for liver cirrhosis. -Benzoate and phenylacetate are pharmacologic agents used for the treatment of hyperammonemia in liver cirrhosis and inherited urea cycle enzyme deficiency. -Sodium benzoate: combines with endogenous glycine to from hippuric acid that is cleared by the kidneys. -Phenyl acetate: conjugates with glutamine to form phenylacetyl-glutamine, which is excreted in urine. -Arginine: supplies urea cycle with ornithine and N-acetylglutamate and is effective in treatment of hyperammonemia due to defects of urea cycle (except in patients with arginase deficiency).

Fate of Ammonia: Detoxified into Urea -Ammonia is a hazardous waste. It is neurotoxic even in low concentrations, and therefore, has to be dispose off quickly. -Human turn it into non-toxic water soluble and therefore, easily excretable urea. -Urea is produced by the liver, and then transported in the blood to kidneys for excretion in the urine. -

-A portion of urea diffuses from blood into intestine and is cleaved to carbon dioxide and ammonia, a reaction catalyzed by bacterial enzyme urease. This ammonia is partly lost in feces or reabsorbed into the blood. -1 g of urea is formed from 3 g of dietary protein. -Blood urea level is measures as blood urea nitrogen (BUN). 250-700 µM/l. BUN rises sharply in renal failure. This condition is called Uremia.

a. In the mitochondria: -Carbon dioxide and ammonia are converted to carbamoyl phosphate which is condensed with ornithine to form citrulline. The deficiency in the ornithine transcarbamoylase results in hyperammonemia and high level of urine orotate as the carbamoyl phosphate is a utilized for the formation of orotic acid. -The formation of carbamoyl phosphate is driven by the cleavage of 2 molecules of ATP. This reaction is catalyzed by carbamoyl phosphate synthetase I (CPS I). There is also a cytoplasmic form of this enzyme (CPS II) involved in pyrimidine synthesis.

-Carbamoyl phosphate synthetase I catalyzes the rate limiting step in the cycle and the enzyme has a requirement for N-acetyl glutamate. -Ornithine is regenerated in the cytosol with each turn of urea cycle and transported back to mitochondria and can be used for another cycle. When cell require additional ornithine it is synthesized from glucose. -The reaction product citrulline is transported to the cytosol.

REMOVAL OF NITROGEN FROM THE AA -Removing the α-amino group from the amino acid is an obligatory step in the catabolism of amino acids as α-amino group protects the amino acids from oxidative breakdown. Once removed, the nitrogen can be incorporated into the other compounds or excreted, with carbon skeleton being metabolized.

-Catabolism of most of the amino acids is via two-step process: transamination, and oxidative deamination- Reactions that ultimately provide ammonia and aspartate, the two sources of urea nitrogen.

see slide 18 b in the cytoplasm

-Citrulline combines with aspartate to form argininosuccinate, a reaction catalyzed by argininosuccinate synthetase. Reaction is driven by hydrolysis of ATP to AMP and inorganic pyrophosphate. -Argininosuccinate is cleaved to form arginine and fumarate, catalyzed by argininosuccinate lyase. -Arginine is cleaved to urea and regenerate ornithine with the help of arginase, an enzyme located primarily in liver and is inhibited by ornithine. -The Krebs bicycle, indicating the common steps between the TCA and urea cycles.

Urea Cycle Disorders: INCREASE IN AMMONIA IN BLOOD

-Failure of urea cycle which can be hereditary (deficiency of urea cycle enzymes (carbamoyl phosphate synthetase I,ornithine transcarbamoylase, arginosuccinate synthetase, and argininosuccinate lyase) or acquired (severe liver diseases due to viral hepatitis, ischemia, or hepatotoxin etc.) causes hyperammonemia. [Note: Unlike deficiencies of other enzymes in urea cycle, arginase deficiency does not result in severe hyperammonemia, since it has a peripheral isozyme which is induced when liver enzyme is dysfunctional and also because arginine containing the waste nitrogen can be excreted into the urine). -Hyperammonemia causes central nervous system dysfunction. The prevalence of urea cycle disorders is estimated at 1 case per 30,000 live births in United States. -Hyperammonemia causes functional disturbances of central nervous system leading to brain damage, coma, and /or death. It plays significant role in development of diseases as cirrhosis, liver encephalopathy, alcohol intoxication and Alzheimer's disease.

SOURCES OF AMMONIA -From AA: form ammonia from AA by transdeamination-the linking of aminotransferase and glutamate dehydrogenase reactions. -From glutamine: The kidneys form ammonia from glutamine by the actions of renal glutaminase and glutamate dehydrogenase. Most of this ammonia is excreted into the urine as NH4+, which provides an important mechanism for maintaining the body's acid-base balance. Ammonia is also obtained from the hydrolysis of glutamine by intestinal glutaminase. The intestinal mucosal cells obtain glutamine either from the blood or from digestion of dietary protein.

-From bacterial action in the intestine: Ammonia is formed from urea by the action of bacterial urease in the lumen of the intestine. This ammonia is absorbed from the intestine by way of the portal vein and is almost quantitatively removed by the liver and converted to urea. -From amines: Amines obtained from the diet, and monoamines that serve as hormones or neurotransmitters, give rise to ammonia by the action of amine oxidases. -From purines and pyrimidines: In the catabolism of purines and pyrimidines, amino groups attached to the rings are released as ammonia.

Transport of Ammonia in the Circulation. Although ammonia is constantly produced in tissues, very low levels of ammonia are maintained in blood because of its toxicity. Low levels are maintained by: (i) rapid removal of blood ammonia by liver and (ii) most tissues release amino acid nitrogen in the form of glutamine or alanine, rather than free ammonia.

-Glutamine: Acts as the carrier of NH4.The ATP-dependent formation of glutamine from glutamate and ammonia by glutamine synthetase occurs primarily in the muscle and liver but also important in nervous system. Glutamine is transported from blood to liver where it is converted into glutamate and free ammonia. -Alanine: Second transport mechanism of NH4 involves transamination of pyruvate to form alanine. Alanine is transported via blood to the liver where it gives pyruvate again by transamination. The pathway of gluconeogenesis can use the pyruvate to form glucose which can enter the blood and be used by the muscle-a pathway called glucose-alanine cycle.

The UREA Cycle: Urea is Synthesized

-The urea cycle converts ammonia to urea, a less toxic molecule. One nitrogen atoms of urea is supplied by ammonia and the other is from aspartate. The carbon and oxygen of the urea are derived from CO2. -Citrulline is transported across the inner mitochondrial membrane by a carrier for neutral amino acids. -Ornithine is transported in exchange for H+ or citrulline. -Fumarate produced in urea cycle is hydrated to malate which can transported to mitochondria via malate shuttle and reenter TCA cycle. Alternately, cytosolic malate can be oxidized to oxaloacetate, which can be converted to aspartate or glucose.

Summary of the Urea Cycle 1. Mitochondrial carbamyl phosphate synthetase I catalyzes the ATP-dependent conversion of HCO3- and NH4+ to the energy rich, mixed anhydride carbamyl phosphate. (Carbamyl phoshate synthetase I should be distinguished from the cytosolic form of the enzyme, carbamyl phosphatase II, which is involved in pyrimidine biosynthesis.)Two molecules of ATP are converted to ADP in this process.

2. Ornithine transcarbamylase, also known as ornithine carbamyltransferase, is associated with carbamyl phosphate synthetase I in the mitochondrial matrix. It catalyzes the nucleophilic addition of ornithine ( which is produced in the cytosol and transported into the mitochondrial matrix by a specific transport protein in the inner mitochondrial membrane) to the carbonyl group of carbamyl phosphate to produce citrulline. 3. After citrulline has been transported to the cytosol, it condenses with aspartate to form arginosuccinate in an ATP-dependent reaction catalyzed by arginosuccinate synthestase.

Urea Cycle In mitochondria 1) HCO3- + NH4+--> CARBAMOYL PHOSPHATE(HIGH ENERGY BOND) BY CARBAMOYL PHOSPHATE SYNTHETASE I(RATE-LIMITING ENZYME) AND ATP IS USED. 2) ORNITHINE + carbamoyl phosphate--> citrulline by ornithine transcarbamoylase CITRULLINE IS EXPORTED INTO THE CYTOSOL

3) citrulline+aspartate--> argininosuccinate by argininosuccinate synthetase and ATP is used. 4) argininosuccinate--> fumarate(CONNECTED WITH TCA CYCLE) + arginine by argininosuccinate lyase 5) arginine--> ornithine by arginase ORNITHINE TRANSPORTED BACK INTO THE MITOCHONDRIA.

-All of the reactions are irreversible except glutamate dehydrogenase (GDH). -Only the dehydratase reactions, which produce NH4+ from serine and threonine, require pyridoxal phosphate as a cofactor. -The reactions that are shown occurring in the muscle or the gut can all occur in the liver, where the NH4+ generated can be converted to urea.

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LOOK AT SLIDES HIGHLIGHTED

LOOK AT SLIDES HIGHLIGHTED.

Regulation of Urea Cycle -N-acetylglutamate is the activator of carbamoyl phosphate synthetase I (CPSI), the rate limiting step of urea cycle. Arginine stimulates the synthesis of N-acetyl glutamate (a positive allosteric effector of N-acetylglutamate synthetase), which activates CPSI. Therefore, the intrahepatic concentration of N-acetylglutamate increases after ingestion of a protein-rich meal, which provides both the substrate (glutamate) and regulator of N-acetylglutamate synthesis. This leads to an increased rate of urea synthesis.

Figure 19.16 Formation and degradation of N-acetylglutamate, an allosteric activator of carbamoyl phosphate synthetase I.

.Mechanism of action of Aminotransferases. All aminotransferases require the coenzyme pyridoxal phosphate which is covalently linked to the ε-amino group of a specific lysine residue at the active site of the enzyme. Aminotransferase act by transferring the amino group of an amino acid to the pyridoxal part of the coenzyme to generate pyridoxamine phosphate which react with an α-keto acid to form an amino acid, and generating the original aldehyde form of the coenzyme.

For most of the aminotransferases the equilibrium constant is near one. This allows the reaction to go in both directions, 1. amino acid degradation (after consumption of protein-rich meal) and, 2. amino acid biosynthesis (when the supply of amino acids from the diet is not adequate)

Role of Glutamate in Urea Production. Glutamate dehydrogenase

Glutamate collects nitrogen from other amino acids by transamination reactions. This nitrogen can be released as NH4+ by glutamate dehydrogenase (GDH). NH4+ is also produced by other reactions. NH4+ provides one of the nitrogens for urea synthesis. The other nitrogen comes from aspartate and is obtained from glutamate by transamination of oxaloacetate.