1. UREA CYCLE Urea is the major disposal form of amino groups derived from amino acids, and accounts for about 90% of the nitrogen- containing components of urine. One nitrogen of the urea molecule is supplied by free ammonia, and the other nitrogen by aspartate. The carbon and oxygen of urea are derived from CO 2. Urea is produced by the liver, and then is transported in the blood to the kidneys for excretion in the urine.

2. Reactions of the cycle 1. Formation of carbamoyl phosphate: Formation of carbamoyl phosphate by carbamoyl phosphate synthetase I is driven by cleavage of two molecules of ATP. 2. Formation of citrulline: The carbamoyl portion of carbamoyl phosphate is transferred to ornithine by ornithine transcarbamoylase (OTC) as the high-energy phosphate is released as P i. The reaction product, citrulline, is transported to the cytosol. 3. Synthesis of argininosuccinate: Argininosuccinate synthetase combines citrulline with aspartate to form argininosuccinate. The α-amino group of aspartate provides the second nitrogen that is incorporated into urea. The formation of argininosuccinate is driven by the cleavage of ATP to adenosine monophosphate (AMP) and pyrophosphate. This is the third and final molecule of ATP consumed in the formation of urea. 4. Cleavage of argininosuccinate: The arginine formed by this reaction serves as the immediate precursor of urea. 5. Cleavage of arginine to ornithine and urea: Arginase cleaves arginine to ornithine and urea, and occurs almost exclusively in the liver. Kidney, can synthesize arginine by these reactions, but only the liver can cleave arginine and, thereby, synthesize urea.

4. Overall stoichiometry of the urea cycle Aspartate + NH 3 + CO 2 + 3ATP + H 2 O → urea + fumarate + 2ADP + AMP + 2P i + PP i Four high-energy phosphate bonds are consumed in the synthesis of each molecule of urea; therefore, the synthesis of urea is irreversible, with a large, negative ΔG. One nitrogen of the urea molecule is supplied by free NH 3, and the other nitrogen by aspartate. Glutamate is the immediate precursor of both ammonia and aspartate nitrogen.

5. Regulation of the urea cycle N-Acetylglutamate is an essential activator for carbamoyl phosphate synthetase I – the rate-limiting step in the urea cycle. N-Acetylglutamate is synthesized from acetyl coenzyme A and glutamate by N-acetylglutamate synthase, in a reaction for which arginine is an activator. Intrahepatic concentration of N-acetylglutamate increases after ingestion of a protein-rich meal, which provides both a substrate (glutamate) and the regulator of N- acetylglutamate synthesis. This leads to an increased rate of urea synthesis.

6. 1. From glutamine: The kidneys generate ammonia from glutamine by the actions of renal glutaminase and glutamate dehydrogenase. Most of this amonia is excreted into the urine as NH 4 +, which provides an important mechanism for maintaining the body’s acid-base balance through the excretion of protons. 2. From bacterial action in the intestine: Ammonia is formed from urea by the action of bacterial urease in the lumen of the intestine. 3. From amines: Amines obtained from the diet, and monoamines that serve as hormones or neurotransmitters, give rise to ammonia by the action of amine oxidase. 4. From purines and pyrimidines: In the catabolism of purines and pyrimidines, amino groups attached to the rings are released as ammonia. Sources of ammonia

7. Transport of ammonia in the circulation Although ammonia is constantly produced in the tissues, it is present at very low levels in blood. This is due both to the rapid removal of blood ammonia by the liver, and the fact that many tissues, particularly muscle, release amino acid nitrogen in the form of glutamine or alanine, rather than as free ammonia. 1. Urea: Formation of urea in the liver is quantitatively the most important disposal route for ammonia. Urea travels in the blood from the liver to the kidneys, where it passes into the glomerular filtrate. 2. Glutamine: This amide of glutamic acid provides a nontoxic storage and transport form of ammonia. The ATP-requiring formation of glutamine from glutamate and ammonia by glutamine synthetase occurs primarily in the muscle and liver. Circulating glutamine is removed by the liver and the kidneys and deaminated by glutaminase.

8. HYPERAMMONEMIA The levels of serum ammonia are normally low (5-35 µmol/L). However, when liver function is compromised, due either to genetic defects of the urea cycle or liver disease, blood levels can rise above 1,000 µmol/L. Such hyperammonemia is a medical emergency, because ammonia has a direct neurotoxic effect on the CNS (tremors, slurring of speech, somnolence, vomiting, cerebral edema, and blurring of vision). At high concentrations, ammonia can cause coma and death. 1.Acquired hyperammonemia: Liver disease is a common cause of hyperammonemia in adults (viral hepatitis or hepatotoxins such as alcohol, cirrhosis of the liver). As a result, portal blood is shunted directly into the systemic circulation and does not have access to the liver. The conversion of ammonia to urea is, severely impaired, leading to elevated levels of ammonia. 2.Congenital hyperammonemia: Genetic deficiencies of each of the five enzymes of the urea cycle have been described, with an overall prevalence estimated to be 1:25,000 live births. Ornithine transcarbamoylase deficiency, is the most common of these disorders. In each case, the failure to synthesize urea leads to hyperammonemia during the first weeks following birth. Historically, urea cycle defects had high morbidity (neurological manifestations) and mortality. Treatment included restriction of dietary protein in the presence of sufficient calories to prevent catabolism.

9. BIOSYNTHESIS OF NONESSENTIAL AMINO ADICS Nonessential amino acids are synthesized from intermediates of metabolism or, as in the case of tyrosine and cysteine, from the essential amino acids phenylalanine and methionine, respectively. Synthesis from α-keto acids Alanine, aspartate, and glutamate are synthesized by transfer of an amino group to the α-keto acids pyruvate, oxaloacetate, and α-ketoglutarate, respectively by transamination reactions. Synthesis by amidation 1. Glutamine: Is formed from glutamate by glutamine synthetase. 2. Asparagine: Is formed from aspartate by asparagine synthetase, using glutamine as the amide donor.

10. 1.Serine: Arises from 3-phosphoglycerate which is first oxidized to 3-phosphopyruvate, and then transaminated to 3-phosphoserine. Serine is formed by hydrolysis of the phosphate ester. Serine can also be formed from glycine through transfer of a hydroxymethyl group by serine hydroxymethyl transferase using N 5,N 10 - methylene-THF as the one carbon donor. 2.Glycine: Is synthesized from serine by removal of a hydroxymethyl group, also by serine hydroxymethyl transferase. THF is the one carbon acceptor. 3.Proline: Glutamate is converted to proline by cyclization and reduction reactions. 4.Cysteine: Is synthesized by two consecutive reactions in which homocysteine combines with serine, forming cystathionine that is hydrolyzed to α-ketobutyrate and cysteine. Because methionine is an essential amino acid, cysteine synthesis can be sustained only if the dietary intake of methionine is adequate. 5.Tyrosine: Is formed from phenylalanine by phenylalanine hydroxylase. Tyrosine, like cysteine, is formed from an essential amino acid and is, therefore, nonessential only in the presence of adequate dietary phenylalanine. S-Adenosylhomocysteine (SAH) L -Methionine S-Adenosylmethionine (SAM) L -Homocysteine Cystathionine L -Cysteine

12. Leucine Lysine Isoleucine Phenyl- alanine Tryptophan Histidine Methionine Threonine Valine TyrosineAlanine Arginine Asparagine Aspartate Cysteine Glutamate Glutamine Glycine Proline Serine Ketogeni c Glucogenic and Ketogenic Glucogenic Essential Nonessential GLUCOGENIC AMINO ACIDS Amino acids whose catabolism yields pyruvate or one of the intermediates of the citric acid cycle are termed glucogenic. These intermediates are substrates for gluconeogenesis and can give rise to the net formation of glucose in the liver and kidney. KETOGENIC AMINO ACIDS Amino acids whose catabolism yields either acetoacetate or one of its precursors (acetyl CoA or acetoacetyl CoA). Leucine and lysine are the only exclusively ketogenic amino acids found in proteins.

13. Amino acids that form oxaloacetate Asparagine is hydrolyzed by asparaginase, liberating ammonia and aspartate. Aspartate loses its amino group by transamination to form oxaloacetate. Amino acids that form α-ketoglutarate via glutamate 1. Glutamine: Is converted to glutamate and ammonia by the enzyme glutaminase. Glutamate is converted to α-ketoglutarate by transamination, or through oxidative deamination by glutamate dehydrogenase. 2. Proline: Is oxidized to glutamate. 3. Arginine: Is cleaved by arginase to produce ornithine. Ornithine is subsequently converted to α-ketoglutarate. 4. Histidine: Is oxidatively deaminated by histidase to urocanic acid, which subsequently forms N-formiminoglutamate and finally glutamate.

14. Amino acids that form pyruvate 1. Alanine: This amino acid loses its amino group by reversible transamination to form pyruvate. 2. Serine: This amino acid can be converted to glucine and N 5, N 10 – methylene tetrahydrofolate or can also be converted to pyruvate by serine dehydratase. 3. Glycine: Can be converted to serine. 4. Cysteine undergoes desulfuration to yield pyruvate. 5. Threonine: Is converted to pyruvate or to α-ketobutyrate, which forms succinyl CoA. Amino acids that form fumarate Phenylalanine and tyrosine: Hydroxylation of phenylalanine produces tyrosine. This reaction, catalyzed by phenylalanine hydroxylase. The metabolism Phe and Tyr lead ultimately to the formation fumarate and acetoacetate. Phenylalanine and tyrosine are, therefore, both glucogenic and ketogenic.

15. Amino acids that form succinyl: Methionine Resynthesis of methionine: Homocysteine accepts a methyl group from N 5 - methyltetrahydrofolate (N 5 -methyl-THF) in a reaction requiring methylcobalamin, a coenzyme derived from vitamin B 12. Valine and isoleucine: Are branched-chain amino acids that generate propionyl CoA, which is converted to succinyl CoA by biotin- and vitamin B 12 -requiring reactions. Threonine: Is dehydrated to α-ketobutyrate, which is converted to propionyl CoA and then to succinyl CoA. Threonine can also be converted to pyruvate. CH 3 S + CH 2 HCNH 3 + COO – Adenosine – S CH 2 HCNH 3 + COO – SH CH 2 HCNH 3 + COO – CH 2 CH 2 – S – CH 2 HCNH 3 + COO – S-Adenosylhomocysteine (SAH) H2OH2O Adenosine S-Adenosyl- methionine synthetase L -Methionine ATP P i + PP i Mg 2+ S-Adenosylmethionine (SAM) Methyl acceptors Methylated products Methyltransferases L -Homocysteine B6B6 Cystathionine β-synthase L -Serine H2OH2O Cystathionine B6B6 H2OH2O α-Ketobutyrate + NH 4 + L -Cysteine H H N CH 3 N CH 2 N – H COO – CH 2 S H – C – NH 3 + CH 3 H H N N CH 2 N – H H + + N5-Methyltetrahydrofolate Methionine synthase Methylcobalamin (Methyl-B 12 ) L-Methionine Tetrahydrofolate There are two major disposal pathways for homocysteine. Conversion to methionine requires folate and vitamin B 12 -derived coenzymes, and is a remethylation process. The formation of cysteine requires vitamin B 6 (pyridoxine), and is a transsulfuration process. There are two major disposal pathways for homocysteine. Conversion to methionine requires folate and vitamin B 12 -derived coenzymes, and is a remethylation process. The formation of cysteine requires vitamin B 6 (pyridoxine), and is a transsulfuration process.

16. Amino acids that form acetyl CoA or acetoacetyl Coa Leucine, isoleucine, lysine, and tryptophan form acetyl CoA or acetoacetyl CoA directly, without pyruvate. 1. Leucine: This amino acid is exclusively ketogenic in its catabolism, forming acetyl CoA and acetoacetate. 2. Isoleucine: Is both ketogenic and glucogenic, because its metabolism yields acetyl CoA and propionyl CoA. 3. Lysine: An exclusively ketogenic amino acid, this amino acid is unusual in that neither of its amino groups undergoes transamination as the first step in catabolism. Lysine is ultimately converted to acetoacetyl CoA. 4. Tryptophan: Is both glucogenic and ketogenic because its metabolism yields alanine and acetoacetyl CoA.

17. Catabolism of the branched-chain amino acids The branched-chain amino acids, isoleucine, leucine, and valine, are essential amino acids. In contrast to other amino acids, they are metabolized primarily by the peripheral tissues (particularly muscle), rather than by the liver. 1.Transamination: Removal of the amino groups is catalyzed by vitamin B 6 -requiring enzyme, branched-chain amino acid aminotransferase. 2.Oxidative decarboxylation: Removal of the carboxyl group of the α-keto acids multienzyme complex, branched-chain α-keto acid dehydrogenase complex. This complex uses thiamine pyrophosphate, lipoic acid, FAD, NAD +, and CoA as its coenzymes. An inherited deficiency of branched-chain α-keto acid dehydrogenase results in accumulation of the branched-chain α- keto acid substrates in the urine. Their sweet odor prompted the name maple syrup urine disease. 3.Dehydrogenation. 4.End products: The catabolism of isoleucine yields acetyl CoA and succinyl CoA. Valine yields succinyl CoA and is glucogenic. Leucine is ketogenic, being metabolized to acetyl CoA.

18. FOLIC ACID Folic acid plays a key role in one- carbon metabolism, is essential for the biosynthesis of several compounds. Tetrahydrofolate (reduced folate) receives one-carbon fragments from donors such as serine, glycine, and histidine and transfers them to intermediates in the synthesis of amino acids, purines, and thymidine monophosphate (TMP).

19. COBALAMIN (BITAMIN B 12 ) Vitamin B 12 is required in humans for two essential enzymatic reactions: the remethylation of homocysteine to methionine and the isomerization of methylmalonyl coenzyme A. Cobalamin contains a corrin ring system that differs from the porphyrins. Cobalt is held in the center of the corrin ring by four coordination bonds from the nitrogens of the pyrrole groups. Vitamin B 12 is synthesized only by microorganisms; it is not present in plants. Cobalamin is present in appreciable amounts in liver, whole milk, eggs, oysters, fresh shrimp, pork, and chicken.

20. 1. Synthesis of SAM: Methionine condenses with adenosine triphosphate (ATP), forming SAM – a high-energy compound that is unusual in that it contains no phosphate. 2. Activated methyl group: The methyl group attached to the tertiary sulfur in SAM is “activated”, and can be transferred to a variety of acceptor molecules. CH 3 S + CH 2 HCNH 3 + COO – Adenosine – S-Adenosyl- methionine synthetase L -Methionine ATP P i + PP i Mg 2+ S-Adenosylmethionine (SAM) Methyl acceptors Methylated products Methyltransferases

22. CREATINE Creatine phosphate found in muscle, is a high- energy compound that provides a small but rapidly mobilized reserve of high-energy phosphates that can be reversibly transferred to ADP to maintain the intracellular level of ATP during the first few minutes of intense muscular contraction. 1. Synthesis: Creatine is synthesized from glycine and the guanidino group of arginine, plus a methyl group from SAM. Creatine is reversibly phosphorylated to creatine phosphate by creatine kinase, using ATP as the phosphate donor. 2. Degradation: Creatine and creatine phosphate spontaneously cyclize at a slow but constant rate to form creatinine, which is excreted in the urine. The amount of creatinine excreted is proportional to the total creatine phosphate content of the body, and thus can be used to estimate muscle mass. Any rise in blood creatinine is a sensitive indicator of kidney malfunction, because creatinine normally is rapidly removed from the blood and excreted.

23. The plasma levels of creatine kinase (CK) are commonly determined in the diagnosis of myocardial infarction. They are particularly useful when the electrocardiogram is difficult to interpret, such as when there have been previous episodes of heart disease. Creatine kinase (CK) occurs as three isoenzymes. Each isoenzyme is a dimer composed of two polypeptides (called B and M subunits) associated in one of three combinations: CK1 = BB, CK2 = MB, and CK3 = MM. CK isoenzyme shows a characteristic electrophoretic mobility. Myocardial muscle is the only tissue that contains more than 5 % of the total CK activity as the CK2 (MB) isoenzyme. Appearance of this hybrid isoenzyme in plasma is virtually specific for infarction of the myocardium. Following an acute MI, this isoenzyme appears approximately 4-8 hours following onset of chest pain, reaches a peak of activity at approximately 24 hours, and returns to baseline after 48-72 hours.