1. AMINO ACID DEGRADATION AND SYNTHESIS Essential and nonessential aminoacids Glucogenic and ketogenic amino acids One-Carbon Metabolism Degradation of the carbon skeletons of amino acids Biosynthesis of nonessential amino acids OBJECTIVES—LEARNING OBJECTIVES HANDOUT Dr O. Morebise

2. AMINO ACID DEGRADATION The catabolism of the amino acids found in proteins involves the removal of α- amino groups, followed by the breakdown of the resulting carbon skeletons. These pathways converge to form seven intermediate products : Oxaloacetate, α-ketoglutarate, pyruvate, fumarate, succinyl CoA, acetyl CoA, and acetoacetate. These products directly enter the pathways of intermediary metabolism, resulting either in the synthesis of glucose or lipid, or in the production of energy through their oxidation to CO 2 and water by the citric acid cycle. Note: Intermediary metabolism: The sum of all metabolic reactions between uptake of foodstuffs and formation of excretory products.

3. Essential and nonessential amino acids Essential amino acids are those that cannot be synthesized by the body and must, therefore, be obtained from the diet in order for normal protein synthesis to occur. They include : Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, and Valine. Nonessential amino acids are those that can be synthesized from the intermediates of metabolism or, as in the case of cysteine and tyrosine, from the essential amino acids methionine and phenyl alanine, respectively. The nonessential amino acids include: alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, proline, serine, and tyrosine. Arginine and histidine are considered semi-essential because they are synthesized at rates inadequate to support growth in children and individuals recovering from wasting diseases. As long as sufficient amounts of essential amino acids are present in the diets, the remaining amino acids can be formed through transamination and other reactions.

4. Glucogenic and Ketogenic amino acids Amino acids are classified as glucogenic or ketogenic based on which of the seven intermediates are produced during their catabolism. Amino acids that form pyruvate or intermediates of the TCA cycle are glucogenic (glycogenic); i.e., they provide carbon for the synthesis of glucose. Amino acids that form acetyl CoA, acetoacetyl CoA, or acetoacetate directly without passing through pyruvate are termed ketogenic; i.e., they form ketone bodies. Leucine and lysine are the only exclusively ketogenic amino acids found in proteins.

5. Alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, methionine, proline, serine, threonine, and valine are purely glucogenic; Isoleucine, phenylalanine, tryptophan, and tyrosine are both glucogenic and ketogenic; Glucogenic amino acids are the major carbon source for gluconeogenesis when glucose levels are low.

6. AMINO ACIDS THAT FORM OXALOACETATE Asparagine is hydrolyzed by asparaginase, liberating ammonia and aspartate. Aspartate loses its amino group by transamination to form oxaloacetate. Note: Some rapidly dividing leukemic cells are unable to synthesize sufficient asparagine to support their growth. This makes asparagine an essential amino acid for these cells, which therefore require asparagine from the blood. Asparaginase, which hydrolyzes asparagine to aspartate, can be administered systemically to treat leukemic patients. Asparaginase lowers the level of asparagine in the plasma and, therefore, deprives cancer cells of a required nutrient.

7. Amino acids that form α- ketoglutarate Glutamine is converted to glutamate and ammonia by the enzyme glutaminase. Proline is oxidized to glutamate. Glutamate is transaminated or oxidatively deaminated to form α- ketoglutarate. Arginine is cleaved by arginase to produce ornithine. (This reaction occurs primarily in the liver as part of the urea cycle) Ornithine is subsequently converted to α-ketoglutarate. Histidine is oxidatively deaminated by histidase to urocanic acid, which subsequently forms N-formiminoglutamate (FIGlu). FIGlu donates its formimino group to tetrahydrofolate, leaving glutamate, which is converted to α-ketoglutarate. The FIGlu excretion test has been used in diagnosing a deficiency of folic acid. Individuals deficient in folic acid excrete increased amounts of FIGlu in the urine, particularly after ingestion of a large dose of histidine.

8. Figlu HN=CH–NH–CH(COOH)CH 2 CH 2 COOH

10. Amino acids that form pyruvate 1.Alanine is converted to pyruvate by transamination. 2.Serine is converted to glycine and N 5,N 10 - methylenetetrahydrofolate. Serine can also be converted to pyruvate by serine dehydratase (serine dehydrase). 3.Glycine can either be converted to serine by addition of a 1- carbon group from N 5,N 10 -methylenetetrahydrofolate, or oxidized to CO 2 and NH 4 +. 4.Cystine is reduced to cysteine, using NADH + H + as a reductant. Cysteine undergoes desulfuration to yield pyruvate. 5.Threonine is converted to pyruvate or to α-ketobutyrate, which forms succinyl CoA.

11. Catabolic pathways for alanine, glycine, serine, cysteine, tryptophan, and threonine

12. Amino acids that form fumarate Phenylalanine and tyrosine: hydroxylation of phenylalanine leads to the formation of tyrosine. The reaction is catalyzed by phenylalanine hydroxylase, and is the first reaction in the catabolism of phenylalanine. Catabolism of phenylalanine and tyrosine leads to the formation of fumarate and acetoacetate. Phenylalanine and tyrosine are, therefore, both glucogenic and ketogenic. Inherited deficiencies in the enzymes of phenylalanine and tyrosine metabolism lead to the diseases phenylketonuria, and alkaptonuria, and the condition of albinism.

13. Benzene ring + 2OH = catechol Catechol + amine NH3 = catecholamine

14. Amino acids that form succinyl CoA— methionine, valine, isoleucine, and threonine METHIONINE Methionine is converted to S- adenosylmethionine (SAM), the major methyl-group donor in one carbon metabolism. Methionine is also the source of homocysteine—a metabolite associated with atherosclerotic vascular disease.

15. More stable

16. The methyl group attached to the tertiary sulfur in SAM is “activated”, and can be transferred to a variety of acceptor molecules, such as ethanolamine in the synthesis of choline. The methyl group is usually transferred to oxygen or nitrogen atoms, but sometimes to carbon atoms. After donation of the methyl group, SAM is hydrolyzed to homocysteine. Homocysteine may be remethylated to methionine (the reaction requires methylcobalamin as coenzyme) If methionine stores are adequate, homocysteine may enter the trans- sulfuration pathway, where it is converted to cysteine. Cysteine is not an essential amino acid as long as sufficient methionine is available.

17. Need PLP (B6) Needs PLP (B6) Need Folic acid & B12

18. α-Ketobutyrate is oxidatively decarboxylated to form propionyl CoA which is converted to succinyl CoA.

19. Role of homocysteine in vascular disease Elevated plasma homocysteine levels are an independent cardiovascular risk factor that correlates with the severity of coronary artery disease. Dietary supplementation with folate, vitamin B12, and vitamin B6---the three vitamins involved in the metabolism of homocysteine---leads to a reduction in circulating levels of homocysteine. Patients with homocystinuria (characterized by high levels of homocysteine caused by cystathionine synthase deficiency) experience premature vascular disease, and usually die of myocardial infarction, stroke, or pulmonary embolus. Thus, there is an association of elevated homocysteine with cardiovascular disease.

20. ONE-CARBON METABOLISM Folate reductase reversible Methyl THF reductase Liberates folic homocysteine methyl transferase  Propionyl CoA  Succinyl CoA

21. Other amino acids that form succinyl CoA Valine and isoleucine are degraded to form succinyl CoA. Threonine is converted to α-ketobutyrate, which is converted to propionyl CoA, the precursor of succinyl CoA.

23. Amino acids that form acetyl CoA and acetoacetyl CoA Leucine, isoleucine, lysine, and tryptophan form acetyl CoA or acetoacetyl CoA directly, without pyruvate acting as an intermediate. Phenylalanine and tyrosine also give rise to acetoacetate during their catabolism. Therefore, there are a total of six ketogenic amino acids.

24. 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. These three amino acids have a similar route of catabolism.

25. 1.Transamination :removal of the amino groups of all the three amino acids is catalyzed by a single enzyme, branched-chain α-amino acid aminotransferase. 2. Oxidative decarboxylation : removal of the carboxyl group of the α-keto acids derived from the 3 amino acids is also catalyzed by a single enzyme complex, branched-chain α-keto acid dehydrogenase complex. This complex uses TPP, lipoic acid, FAD, NAD +, and coenzyme A as its coenzymes. An inherited deficiency of this enzyme results in accumulation of the branched-chain keto acid substrates in the urine. Their sweet odor prompted the name maple syrup urine disease.

26. 3. Dehydrogenation : oxidation of the products formed in the above reaction yields α-β-unsaturated acyl CoA derivatives. This reaction is analogous to the dehydrogenation described in the β-oxidation scheme of fatty acid degradation. 4. End products : the catabolism of isoleucine ultimately yields acetyl CoA and succinyl CoA, rendering it both ketogenic and glucogenic. Valine yields succinyl CoA and is glucogenic. Leucine is ketogenic, being metabolized to acetoacetate and acetyl CoA.

27. L-Leucine → α-ketoisocaproate → Isovaleryl CoA → β-methyl crotonyl CoA →→→ Acetoacetyl CoA → Acetyl CoA L-Isoleucine → α-keto-β-methyl valerate → α- methylbutyryl CoA → Tiglyl CoA → Propionyl CoA (+Acetyl CoA) → Methylmalonyl CoA → Succinyl CoA L-Valine → α-ketoisovalerate → Isobutyryl CoA → Methacrylyl CoA → Propionyl CoA → Methylmalonyl CoA → Succinyl CoA

28. Folic acid as single carbon unit carrier Some synthetic pathways require the addition of single carbon groups. These “one-carbon units” can exist in a variety of oxidation states. ------methane, methanol, formaldehyde, formic acid, and carbonic acid. Carbon units can be transferred from carrier compounds such as tetrahydrofolic acid and S-adenosylmethionine (SAM) to specific structures that are being synthesized or modified. The “one-carbon pool” refers to single carbon units attached to this group of carriers.

29. Amino acid metabolism in muscle During fasting, amino acids are released from muscle protein. Some of the amino acids are partially oxidized in muscle and their remaining carbons are converted to alanine and glutamine. Although about 50% of the amino acids released into the blood from muscle are alanine and glutamine, these two amino acids constitute much less than 50% of the total amino acid residues in muscle protein.

30. Branched chain amino acids are oxidized in muscle. -----Some of their carbons are converted to glutamine and alanine before they are released into the blood. Valine and isoleucine produce succinyl CoA, which feeds into the TCA cycle and forms malate, generating energy. Malate can be converted by the malic enzyme to pyruvate, which is transaminated to alanine or oxidatively decarboxylated to acetyl CoA. Malate can also continue around the TCA cycle to α- ketoglutarate, generating additional energy. α-ketoglutarate forms glutamate, which produces glutamine.

31. The alanine released by muscle is also produced by the glucose-alanine cycle, which involves the transport of glucose from the liver to muscle and the return of carbon atoms to the liver as alanine. --Glucose is oxidized in muscle to pyruvate, producing energy. --Pyruvate can be transaminated to alanine, which travels to the liver carrying nitrogen for urea cycle synthesis and carbon for gluconeogenesis.

32. BIOSYNTHESIS OF NONESSENTIAL AMINO ACIDS A. SYNTHESIS FROM KETO ACIDS : 1. Alanine, from Pyruvate 2. Aspartate, from Oxaloacetate 3. Glutamate, from α- ketoglutarate (by transamination or by the reverse of oxidative deamination, catalyzed by glutamate dehydrogenase.

34. Synthesis by amidation 1. Glutamine, from glutamate. The reaction requires hydrolysis of ATP. Enzyme : Glutamine synthetase. 2. Asparagine, from aspartate. Glutamine is the amide donor. The reaction requires hydrolysis of ATP. Enzyme : Asparagine synthetase.

36. Proline Glutamate is converted to proline by cyclization and reduction reactions.

37. Serine, glycine, and cysteine 1. Serine : it arises from 3-phosphoglycerate, an intermediate in glycolysis. Serine can also be formed from glycine through transfer of a hydroxymethyl group. Glycine ↔ serine. Enzyme : serine hydroxymethyltransferase Group carrier : N 5,N 10 -Methylene-tetrahydrofolate. 2. Glycine is synthesized from serine by removal of a hydroxymethyl group, also by serine hydroxymethyltransferase.

39. Cysteine Cysteine is synthesized by two consecutive reactions in which homocysteine combines with serine, forming cystathionine, which, in turn, 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.

40. QUESTION An individual with megaloblastic anemia is found to have a significant folate deficiency. Erythropoiesis is hampered in this man due to his inability to perform which type of enzymatic action? A. Acyl transfer B. Carboxylation C. Methylation D. Hydroxylation E. Decarboxylation