1.10.3 Amino Acid Metabolism

Snapshot

1. Amino acids are the precursors for numerous nitrogen-containing compounds such as heme, physiologically active amines, and glutathione. 2. Excess amino acids are converted to common metabolic intermediates for use as fuels.The first step in amino acid breakdown is removal of the amino group by transamination. 3. Transaminases require pyridoxal phosphate (PLP) and convert amino acids to their corresponding -keto acids. The amino group is transferred to-ketoglutarate to form glutamate, oxaloacetate to form aspartate, or pyruvate to form alanine. 4. Glutamate is subsequently oxidatively deaminated by glutamate dehydrogenase 5. (GDH) to form ammonia and regenerate ketoglutarate. Hyperinsulinism/hyperammonemia (HI/HA),a genetic disease, is caused by a mutation of the GDH gene that decreases GTP’s ability to inhibit GDH. 6. In the urea cycle, amino groups from NH3 and aspartate combine with HCO3 to form urea. This pathway takes place in the liver, partially in the mitochondrion and partially in the cytosol. 7. It begins with the ATP-dependentcondensation of NH3 and HCO3 by carbamoyl phosphate synthetase, an enzyme with a 96-Å-long tunnel connecting its three active sites through which its highly reactive intermediate products are channeled. 8. The resulting carbamoyl phosphate then combines with ornithine to yield citrulline, which combines with aspartate to form argininosuccinate, which in turn is cleaved to fumarate and arginine. 9. The arginine is then hydrolyzed to urea, which is excreted, and ornithine, which reenters the urea cycle. N-

Acetylglutamate regulates the urea cycle by activating carbamoyl phosphate synthetase allosterically. 10. The keto acid products of transamination reactions are degraded to citric acid cycle intermediates or their precursors. 11. The amino acids leucine and lysine are ketogenic in that they are converted only to the ketone body precursors acetyl-

CoA and acetoacetate. 12. The remaining amino acids are, at least in part, glucogenic in that they are converted to the glucose precursors 13. pyruvate, oxaloacetate, _-ketoglutarate, succinyl-CoA, or fumarate. Alanine, cysteine, glycine, serine, and threonineare converted to pyruvate. 14. Serine hydroxymethyltransferase catalyzes the PLP-dependent CC bond cleavage of serine to form glycine. This reaction requires the transfer of a methylene group from N5,N10-methylene-tetrahydrofolate, which the tetrahydrofolate (THF) obtains from the glycine cleavage system, a multienzyme system. Asparagine and aspartate are converted to oxaloacetate. 15. Ketoglutarate is a product of arginine, glutamate, glutamine, histidine, and proline degradation. Methionine, isoleucine, and valine are degraded to succinyl- CoA. Methionine breakdown involves the synthesis of 16. S-adenosylmethionine (SAM), a sulfonium ion that acts as a methyl donor in many biosynthetic reactions.

Hyperhomocysteinemia, a risk factor for cardiovascular disease, cognitive impairment and neural tube defects, is caused by a deficiency in its folate-dependent degradation. 17. Maple syrup urine disease (MSUD) is caused by an inherited defect in branched-chain amino acid degradation.

Branched-chain amino acid degradation pathways contain reactions common to all acyl-CoA oxidations. 18. Tryptophan is degraded to alanine and acetoacetate. 19. Phenylalanine and tyrosine are degraded to fumarate and acetoacetate. Most individuals with the hereditary disease phenylketonuria lack phenylalanine hydroxylase (PAH), which converts phenylalanine to tyrosine.

Unit 1

The degradation of amino acids converts them to citric acid cycle intermediates or their precursors so that they can be metabolized to CO2 and H2O or used in gluconeogenesis (Figure 1.10.31).

 Indeed, oxidative breakdown of amino acids typically accounts for 10 to 15% of the metabolic energy generated by animals.  The 20 “standard” amino acids (the amino acids of proteins) have widely differing carbon skeletons, so their conversions to citric acid cycle intermediates follow correspondingly diverse pathways. The fraction of metabolic energy obtained from amino acids, whether they are derived from dietary protein or from tissue protein, varies greatly with the type of organism and with metabolic conditions. In animals, amino acids undergo oxidative degradation in three different metabolic circumstances:

1. During the normal synthesis and degradation of cellular proteins some amino acids that are released from protein breakdown and are not needed for new protein synthesis undergo oxidative degradation. 2. When a diet is rich in protein and the ingested amino acids exceed the body’s needs for protein synthesis, the surplus is catabolized; amino acids cannot be stored. 3. During starvation or in uncontrolled diabetes mellitus, when carbohydrates are either unavailable or not properly utilized, cellular proteins are used as fuel. Under all these metabolic conditions, amino acids lose their amino groups to form alpha-keto acids, the “carbon skeletons” of amino acids. The alpha-keto acids undergo oxidation to CO2 and H2O or, often more importantly, provide three- and four-carbon units that can be converted by gluconeogenesis into glucose, the fuel for brain, skeletal muscle, and other tissues. (Figure 1.10.31)

One important feature distinguishes amino acid degradation from other catabolic processes described to this point: every amino acid contains an amino group, and the pathways for amino acid degradation therefore include a key step in which the alpha-amino group is separated from the carbon skeleton and shunted into the pathways of amino group metabolism. Amino acid metabolism takes place in two steps: 1. Amino acid deamination 2. Metabolism of the carbon skeleton

Figure 1.10.31. Overview of amino acid catabolism in mammals. The amino groups and the carbon skeleton take separate but interconnected pathways.

Amino acid deamination

 The first reaction in the breakdown of an amino acid is almost always removal of its alpha-amino group with the object of excreting excess nitrogen and degrading the remaining carbon skeleton or converting (Figure 1.10.32).  Amino acids derived from dietary protein are the source of most amino groups. Most amino acids are metabolized in the liver.

Unit 1  Some of the ammonia generated in this process is recycled and used in a variety of biosynthetic pathways; the excess is either excreted directly or converted to urea or uric acid for excretion, depending on the organism.

 Glutamate and glutamine play especially critical roles in nitrogen metabolism, acting as a kind of general collection point for amino groups.  In the cytosol of hepatocytes, amino groups from most amino acids are transferred to alphaketoglutarate to form glutamate, which enters mitochondria and gives up its amino group to form NH4  Excess ammonia generated in most other tissues is converted to the amide nitrogen of glutamine, which passes to the liver, then into liver mitochondria. Glutamine or glutamate or both are present in higher concentrations than other amino acids in most tissues.  In skeletal muscle, excess amino groups are generally transferred to pyruvate to form alanine, another important molecule in the transport of amino groups to the liver. (Figure 1.10.32)

Figure 1.10.32. Amino group catabolism. (a) Overview of catabolism of amino groups (shaded) in vertebrate liver. (b) Excretory forms of nitrogen. Excess NH4 is excreted as ammonia (microbes, bony fishes), urea (most terrestrial vertebrates), or uric acid (birds and terrestrial reptiles). Notice that the carbon atoms of urea and uric acid are highly oxidized; the organism discards carbon only af ter extracting most of its available energy of oxidation.

 Most amino acids are deaminated by transamination, the transfer of their amino group to an alphaketo acid to yield the alpha-keto acid of the original amino acid and a new amino acid, in reactions catalyzed by aminotransferases (Figure 1.10.33).

Aminotransferase Reactions Occur in Two Stages

1. The amino group of an amino acid is transferred to the enzyme, producing the corresponding keto acid and the aminated enzyme.

2. The amino group is transferred to the keto acid acceptor (e.g., _-ketoglutarate), forming the amino acid product (e.g., glutamate) and regenerating the enzyme.

To carry the amino group, aminotransferases require participation of an aldehyde-containing coenzyme, pyridoxal-5’-phosphate (PLP), a derivative of pyridoxine (vitamin B6) Esmond Snell, Alexander Braunstein, and David Metzler demonstrated that the aminotransferase reaction occurs via a Ping Pong Bi Bi mechanism whose two stages consist of three steps each

Stage I: Conversion of an Amino Acid to an alpha-Keto Acid

Step 1.The amino acids nucleophilic amino group attacks the enzyme–PLP Schiff base carbon atom in a transimination (trans-Schiffization) reaction to form an amino acid–PLP Schiff base (aldimine), with concomitant release of the enzyme’s Lys amino group. This Lys is then free to act as a general base at the active site.

Step 2 The amino acid–PLP Schiff base tautomerizes to an alpha-keto acid–PMP Schiff base by the active site Lys–catalyzed removal of the amino acid _ hydrogen and protonation of PLP atom C4’ via a resonance-stabilized carbanion intermediate. This resonance stabilization facilitates the cleavage of the Calpha-H bond. Step 3.The α-keto acid–PMP Schiff base is hydrolyzed to PMP and an alpha -keto acid.

Stage II: Conversion of an alpha-Keto Acid to an Amino Acid

To complete the aminotransferase’s catalytic cycle, the coenzyme must be converted from PMP back to the enzyme–PLP Schiff base. This involves the same three steps as above, but in reverse order: Step 3’ .PMP reacts with an alpha-keto acid to form a Schiff base. Step 2’ .The α -keto acid–PMP Schiff base tautomerizes to form an amino acid–PLP Schiff base. Step 1’ The ε-amino group of the active site Lys residue attacks the amino acid–PLP Schiff base in a transimination reaction to regenerate the active enzyme–PLP Schiff base, with release of the newly formed amino acid. The reaction’s overall stoichiometry therefore is

Unit 1 The amino groups of most amino acids are consequently funneled into the formation of glutamate or aspartate, which are themselves interconverted by glutamate–aspartate aminotransferase:

Figure 1.10.33. The mechanism of PLP-dependent enzyme catalyzed transamination. The f irst stage of the reaction, in which the -amino group of an amino acid is transf erred to PLP yielding an -keto acid and PMP, consists of three steps: (1) transimination; (2) tautomerization, in which the Lys released during the transimination reaction acts as a general acid–base catalyst; and (3) hydrolysis.The second stage of the reaction, in which the amino group of PMP is transf erred to a diff erent -keto acid to yield a new -amino acid and PLP, is essentially the reverse of the f irst stage: Steps 3., 2., and 1. are, respectively, the reverse of Steps 3, 2, and 1.

 An important exception to the foregoing is a group of muscle aminotransferases that accept pyruvate as their alpha-keto acid substrate. The product amino acid, alanine, is released into the bloodstream and transported to the liver, where it undergoes transamination to yield pyruvate for use in gluconeogenesis. The resulting glucose is returned to the muscles, where it is glycolytically degraded to pyruvate. This is the glucose–alanine cycle (Figure 1.10.34).  The amino group ends up in either ammonium ion or aspartate for urea biosynthesis. Evidently, the glucose–alanine cycle functions to transport nitrogen from muscle to liver (Figure 1.10.33).

Figure 1.10.34. The glucose–alanine cycle

Figure 1.10.35. The oxidative deamination of glutamate by glutamate dehydrogenase. This reaction involves the intermediate f ormation of -iminoglutarate.

 Glutamate is oxidatively deaminated in the mitochondrial matrix by glutamate dehydrogenase (GDH).  Oxidation is thought to occur with transfer of a hydride ion from glutamate’s Calpha to NAD (P)+, thereby forming alphaiminoglutarate, which is hydrolyzed to alpha-ketoglutarate and ammonia.  GDH is allosterically inhibited by GTP, NADH, and nonpolar compounds such as palmitoyl-CoA and steroid hormones.  It is activated by ADP, NAD+, and leucine. The ammonia generated by the process is transferred by glutamine.

 Ammonia is quite toxic to animal tissues (we examine some possible reasons for this toxicity later), and the levels present in blood are regulated (Figure 1.10.34).  In many tissues, including the brain, some processes such as nucleotide degradation generate free ammonia.  In most animals much of the free ammonia is converted to a nontoxic compound before export from the extrahepatic tissues into the blood and transport to the liver or kidneys.

For this transport function, glutamate, critical to intracellular amino group metabolism, is supplanted by L-glutamine.  The free ammonia produced in tissues is combined with glutamate to yield glutamine by the action of

glutamine synthetase.

 This reaction requires ATP and occurs in two steps glutamate and

ATP react to form ADP and aglutamyl phosphate intermediate, which then reacts with ammonia to Figure 1. Excess 10.36. Ammonia transport in the form of glutamine. ammonia in tissues is added to glutamate to form produce glutamine and inorganic glutamine, a process catalyzed by glutamine synthetase. Af ter phosphate. transport in the bloodstream, the glutamine enters the liver and  In most terrestrial animals, glutamine NH4 is liberated in mitochondria by the enzyme glutaminase. in excess of that required for biosynthesis is transported in the blood to the intestine, liver, and kidneys for processing. In these tissues, the amide nitrogen is released as ammonium ion in the mitochondria, where the enzyme glutaminase converts glutamine to glutamate and NH4+.

Unit 1  The NH4 from intestine and kidney is transported in the blood to the liver. In the liver, the ammonia from all sources is disposed of by urea synthesis.

Other deamination mechanism:

 Two nonspecific amino acid oxidases, L-amino acid oxidase and D-amino acid oxidase, catalyze the oxidation of L- and  D-amino acids, utilizing FAD as their redox coenzyme [rather than NAD(P)+].The resulting FADH2 is reoxidized by O2.

 D-Amino acid oxidase occurs mainly in kidney. Its function is an enigma since D-amino acids are associated mostly with bacterial cell walls.

Urea Cycle And Nitrogen Excretion

 If not reused for the synthesis of new amino acids or other nitrogenous products, amino groups are channeled into a single excretory end product.  Most aquatic species, such as the bony fishes, are ammonotelic, excreting amino nitrogen as ammonia. The toxic ammonia is simply diluted in the surrounding water.  Terrestrial animals require pathways for nitrogen excretion that minimize toxicity and water loss. Most terrestrial animals are ureotelic, excreting amino nitrogen in the form of urea  Birds and reptiles are uricotelic, excreting amino nitrogen as uric acid.  In ureotelic organisms, the ammonia deposited in the mitochondria of hepatocytes is converted to urea in the urea cycle. This pathway was discovered in 1932 by Hans Krebs. Urea production occurs almost exclusively in the liver and is the fate of most of the ammonia channeled there.

Figure 1.10.37. Nitrogen-acquiring reactions in the synthesis of urea. The urea nitrogens are acquired in two reactions, each requiring ATP. (a) In the reaction catalyzed by carbamoyl phosphate synthetase I, the f irst nitrogen enters f rom ammonia. The terminal phosphate groups of two molecules of ATP are used to form one molecule of carbamoyl phosphate. In other words, this reaction has two activation steps ( 1 and 3 ). (b) In the reaction catalyzed by argininosuccinate synthetase, the second nitrogen enters f rom aspartate. The ureido oxygen of citrulline is activated by the addition of AMP in step 1 ; this sets up the addition of aspartate in step 2 , with AMP (including the ureido oxygen) as the leaving group.

Unit 1  The urea cycle begins inside liver mitochondria, butthree of the subsequent steps take place in the cytosol; the cycle thus spans two cellular compartments.  The mechanism of action is as discussed below:  A-Carbamoyl Phosphate Synthetase 1- It catalyzes the condensation and activation of NH3 and to form carbamoyl phosphate, the first of the cycle’s two nitrogen-containing substrates, with the concomitant hydrolysis of two ATPs. The reaction catalyzed by CPS I involves three steps: 1. Activation of by ATP to form carboxyphosphate and ADP. 2. Nucleophilic attack of NH3 on carboxyphosphate, displacing the phosphate to form carbamate and Pi. 3. Phosphorylation of carbamate by the second ATP to form carbamoyl phosphate and ADP. B-Ornithine Transcarboxylase: It transfers the carbamoyl group of carbamoyl phosphate to ornithine, yielding citrulline. The reaction occurs in the mitochondrion so that ornithine, which is produced in the cytosol, must enter the mitochondrion via a specific transport system. The subsequent three steps take place in the cytosol (Figure 1.10.37a). C-Argininosuccinate Synthetase-Urea’s second nitrogen atom is introduced in the urea cycle’s third reaction by the condensation of citrulline’s ureido group with an aspartate amino group by argininosuccinate synthetase. The ureido oxygen atom is activated as a leaving group through formation of a citrullyl–AMP intermediate, which is subsequently displaced by the aspartate amino group. D. Argininosuccinase- It catalyzed elimination of arginine from the aspartate carbon skeleton forming fumarate. Arginine is urea’s immediate precursor. The fumarate produced in the argininosuccinase reaction reacts via the fumarase and malate dehydrogenase reactions to form oxaloacetate which is then used in gluconeogenesis (Figure 1.10.37b). E. Arginase- The urea cycle’s fifth and final reaction is the arginase catalyzed hydrolysis of arginine to yield urea and regenerate ornithine. Ornithine is then returned to the mitochondrion for another round of the cycle.

 The urea cycle thereby converts two amino groups, one from NH3 and one from aspartate, and a carbon atom from to the relatively nontoxic excretion product urea at the cost of four “high-energy” phosphate bonds (three ATP hydrolyzed to two ADP, two , and PPi, followed by rapid PPi hydrolysis).  This energetic cost, together with that of gluconeogenesis, is supplied by the oxidation of the acetylCoA formed by the breakdown of amino acid carbon skeletons.

Regulation of Urea cycle:

 These changes in demand for urea cycle activity are met over the long term by regulation of the rates of synthesis of the four urea cycle enzymes and carbamoyl phosphate synthetase I in the liver (Figure 1.10.38).  Animals on protein-free diets produce lower levels of urea cycle enzymes. The first enzyme in the pathway, carbamoyl phosphate synthetase I, is allosterically activated by N-acetylglutamate, which is synthesized from acetyl- CoA and glutamate by N-acetylglutamate synthase. N-acetylglutamate synthase activity in the liver has a purely regulatory function.

Understanding Biochem

-lactamases, which provide bacteria with a bulletproof vest. A beta-lactamase forms a temporary covalent adduct with the carboxyl group of the opened beta-lactam ring, which is immediately hydrolyzed, regenerating active enzyme. One approach to circumventing antibiotic resistance of this type is to synthesize penicillin analogs, such as methicillin, that are poor substrates for –beta actamases. Another approach is to administer along with antibiotics a beta-lactamase inhibitor such as clavulanate or sulbactam.

Unit 1  The steady-state levels of N-acetylglutamate are determined by the concentrations of glutamate and acetyl-CoA

The five enzymes involved are: 1. Carboxypeptidase synthetase 1 2. Ornithine transcarboxylase 3. Argininosuccinate synthetase 4. Argininosyccinase 5. Arginase 6. The yellow circle incates that transport across mitochondria requires special transport system(Figure 1.10.39).

Figure 1.10.38. Synthesis of N-acetylglutamate and its activation of carbamoyl phosphate synthetase I.

Understanding Biochem

Aspirin irreversibly inactivates the cyclooxygenase activity of COX by acetylating a Ser residue and blocking the enzyme’s active site, thus inhibiting the synthesis of prostaglandins and thromboxanes. Ibuprofen, a widely used nonsteroidal anti inflammatory drug, inhibits the same enzyme. The recent discovery that there are two isozymes of COX has led to the development of more precisely targeted NSAIDs with fewer undesirable side effects.

Figure 1.10.39. The urea cycle. Its five enzymes are (1) carbamoyl phosphate synthetase, (2) ornithine transcarbamoylase, (3) argininosuccinate synthetase, (4) argininosuccinase, and (5) arginase.The reactions occur in part in the mitochondrion and in part in the cytosol with ornithine and citrulline being transported across the mitochondrial membrane by specif ic transport systems (yellow circles). One of the urea amino groups (green) originates as the NH3 product of the glutamate dehydrogenase reaction (top).The other amino group (red) is obtained f rom aspartate through the transf er of an amino acid to oxaloacetate via transamination (right).The f umarate product of the argininosuccinase reaction is converted to oxaloacetate for entry into gluconeogenesis via the same reactions that occur in the citric acid cycle but take place in the cytosol (bottom).The A TP utilized in Reactions 1 and 3 of the cycle can be regenerated by oxidative phosphorylation f rom the NAD(P)H produced in the glutamate dehydrogenase (top) and malate dehydrogenase (bottom) reactions.

Unit 1 The degradation of amino acids converts them to citric acid cycle intermediates or their precursors so that they can be metabolized to CO2 and H2O or used in gluconeogenesis. Indeed, oxidative breakdown of amino acids typically accounts for 10 to 15% of the metabolic energy generated by animals. The overall process can be represented as shown in (Figure 1.10.40). There are two main classes of amino acids:

1. Glucogenic amino acids: whose carbon skeletons are degraded to pyruvate, _-ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate and are therefore glucose precursors 2. Ketogenic amino acids, whose carbon skeletons are broken down to acetyl-CoA or acetoacetate and can thus be converted to ketone bodies or fatty acids

Figure 1.10.40. Summary of amino acid catabolism. Amino acids are grouped according to their major degradative end product. Some amino acids are listed more than once because different parts of their carbon skeletons are degraded to diff erent end products. The f igure shows the most important catabolic pathways in vertebrates, but there are minor variations among vertebrate species. Threonine, f or instance, is degraded via at least two diff erent pathways and the importance of a given pathway can vary with the organism and its metabolic conditions. The glucogenic and ketogenic amino acids are also delineated in the f igure, by color shading. Notice that f ive of the amino acids are both glucogenic and ketogenic. The amino acids degraded to pyruvate are also potentially ketogenic. Only two amino acids, leucine and lysine, are exclusively ketogenic.

Degradation of Alanine, cysteine, glycine, serine and threonine to pyruvate:

The carbon skeletons of six amino acids are converted in whole or in part to pyruvate. The pyruvate can then be converted to either acetyl-CoA (a ketone body precursor) or oxaloacetate (a precursor for gluconeogenesis).

The pathway is as follows: 1. Alanine is directly converted to pyruvate by the action of the enzyme alanine aminotransferase. 2. Serine is converted to pyruvate by serine dehydratase (Figure 1.10.41). 3. Glycine is converted to serine by the enzyme serine hydroxymethyltransferase, another PLPcontaining enzyme. This enzyme utilizes N5,N10-methylene-tetrahydrofolate (N5,N10-methylene-

THF) as a cofactor to provide the C1 unit necessary for this conversion 4. Threonine dehydrogenase- producing alpha-amino-alpha-ketobutyrate, which is converted to acetyl-CoA and glycine by alpha-aminoketobutyrate lyase.

Figure 1.10.41. The pathways converting alanine, cysteine, glycine, serine, and threonine to pyruvate. The enzymes involved are (1) alanine aminotransf erase, (2) serine dehydratase, (3) glycine cleavage system, (4) and (5) serine hydroxymethyltransf erase, (6) threonine dehydrogenase, and (7) α-amino-β-ketobutyrate lyase.

 Asparagine is also converted to oxaloacetate in this manner after its hydrolysis to aspartate by L-

asparaginase. The aspartate so formed can be directly converted to oxaloactate by

transamination (Figure 1.10.42).  L-asparaginase is an effective chemotherapeutic agent in the treatment of cancers that must obtain asparagine from the blood, particularly acute lymphoblastic leukemia.The cancerous cells express particularly low levels of the enzyme asparagine synthetase and hence die without an external source of asparagine. However, L-asparaginase treatment may select for cells with increased levels of asparagine synthetase expression, and hence, in these cases, the surviving cancer cells are resistant to this treatment.

Figure 1.10.42. Degradation of asparagine and aspartate to oxaloacetate

Degradation of Arginine, Glutamate, Glutamine, Histidine and proline to alpha-ketoglutarate:

1. Arginine is converted to ornithine by the enzyme arginase by utilizing a water molecule and the subsequent release of urea. 2. Ornithine is then converted to glutamate 5-semialdehyde by the enzyme ornithine deltaaminotransferase. This step involves the conversion of a molecule of alpha-ketoglutarate to glutamate 3. The cyclic structure of proline is opened by oxidation of the carbon most distant from the carboxyl group to create a Schiff base, then hydrolysis of the Schiff base to a linear semialdehyde, glutamate delta-semialdehyde. This reaction is catalyzed by proline oxidase. 4. The conversion of histidine to alpha-ketoglutarate is a more complex process and is done in the following steps (Figure 1.10.43).

Unit 1 5. The reaction is catalyzed by: a) Histidine amino lyase converts histidine to Urocanate. In this process a molecule of ammonia is released. b) Urocanate hydratase catalyses the conversion of urocanate to 4-imidazole-5-propionate utilizing a molecule of water. c) Imidazolone propionate then converts it into N-formiminoglutamate utilizing a molecule of water d) Glutamate formiminotransferase will convert this into glutamate.

Figure 1.10.43. Catabolic pathways for arginine, histidine, glutamate, glutamine, and proline. These amino acids are converted to -ketoglutarate. The numbered steps in the histidine pathway are catalyzed by 1 histidine ammonia lyase, 2 urocanate hydratase, 3 imidazolonepropionase, and 4 glutamate f ormimino transf erase.

Degradation of methionine, isoleucine, threonine and valine to Succinyl-CoA:

 The methionine degradation is a more complex process and involves the following steps; (Figure 1.10.44). o Methionine degradation begins with its reaction with ATP to form S-adenosylmethionine (SAM) o Methylation reaction involving SAM yields S-adenosylhomocysteine in addition to methylated acceptor. o The former product is hydrolyzed to homocysteine and adenosine.

Figure 1.10.44. Conversion of methionine to homocysteine

 The carbon skeletons of methionine, isoleucine, threonine, and valine are degraded by pathways that yield succinyl-CoA, an intermediate of the citric acid cycle.  Methionine donates its methyl group to one of several possible acceptors through Sadenosylmethionine and three of its four remaining carbon atoms are converted to the propionate of propionyl-CoA, a precursor of succinyl-CoA (Figure 1.10.45).  Isoleucine undergoes transamination, followed by oxidative decarboxylation of the resulting alpha- keto acid. The remaining five-carbon skeleton is further oxidized to acetyl-CoA and propionyl-CoA.  Valine undergoes transamination and decarboxylation, then a series of oxidation reactions that convert the remaining four carbons to propionyl-CoA. Some parts of the valine and isoleucine degradative pathways closely parallel steps in fatty acid degradation.  Threonine is also converted in two steps to propionyl-CoA. This is the primary pathway for threonine degradation in humans. This reaction is catalyzed by the enzyme threonine dehydrogenase.

Understanding Biochem

In the human population there are three common variants, or alleles, of the gene encoding apolipoprotein E. The most common, accounting for about 78% of human apoE alleles, is APOE3; alleles APOE4 and APOE2 account for 15% and 7%, respectively. The APOE4 allele is particularly common in humans with Alzheimer’s disease, and the link is highly predictive. Individuals who inherit APOE4 have an increased risk of late-onset Alzheimer’s disease. Those who are homozygous for APOE4 have a 16-fold increased risk of developing the disease; for those who do, the mean age of onset is just under 70 years. For people who inherit two copies of APOE3, by contrast, the mean age of onset of Alzheimer’s disease exceeds 90 years.

Figure 1.10.45. Catabolic pathways for methionine, isoleucine, threonine, and valine. These amino acids are converted to succinyl-CoA; isoleucine also contributes two of its carbon atoms to acetyl-CoA. The pathway of threonine degradation shown here occurs in humans; a pathway f ound in other organisms . The conversion of homocysteine to –ketobutyrate the conversion of propionyl-CoA to succinyl-CoA.

Degradation of leucine and lysine:

There are several pathways for lysine degradation, the one that proceeds via formation of the alphaketoglutarate– lysine adduct saccharopine predominates in mammalian liver. Oxidative decarboxylation of an alpha-keto acid by a multienzyme complex similar to pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. The enzymes involved in the pathway are as follows: (Figure 1.10.46). 1. Saccharophine dehydrogenase 2. Saccharophine dehydrogenase (NAD+, glutamate forming) 3. Aminoadipate semialdehyde dehydrogenase 4. Aminoadipate aminotraansferase (a PLP enzyme) 5. Alpha-keto acid dehydrogenase 6. Glutyrate-CoA dehydrogenase 7. Decarboxylase 8. Enoyl-CoA hydratase 9. Beta-hydoxyacyl=CoA dehydrogenase 10. HMG-CoA lyase

Figure 1.10.46. The pathway of lysine degradation in mammalian liver. The enzymes involved are (1) saccharopine dehydrogenase (NADP, lysine forming), (2) saccharopine dehydrogenase (NAD, glutamate forming), (3) aminoadipate semialdehyde dehydrogenase, (4) Aminoadipate aminotransf erase (a PLP enzyme), (5) -keto acid dehydrogenase, (6) glutaryl- CoA dehydrogenase, (7) decarboxylase, (8) enoyl-CoA hydratase, (9) -hydroxyacylCoA dehydrogenase, (10) HMG-CoA synthase, and (11) HMG-CoA lyase.

Breakdown of leucine:

The branched chain amino acid is degraded by the following enzyme: 1. Branched chain amino-acid amino transferase. This catalyzes the formation of alpha-ketoisocapronic acid (Figure 1.10.47).

Figure 1.10.47. The degradation of the branched-chain amino acids (leucine. The f irst three reactions of each pathway utilize the common enzymes (1) branched-chain amino acid aminotransf erase, (2) branched-chain -keto acid dehydrogenase (BCKDH), and (3) acyl-CoA dehydrogenase. Isoleucine degradation then continues (left) with (4) enoyl-CoA hydratase, (11) methylcrotonyl-CoA carboxylase (a biotin-dependent enzyme), (12) -methylglutaconyl- CoA hydratase, and (13) HMG-CoA lyase to yield acetyl-CoA and acetoacetate.

2. Branched chain alpha-keto acid dehydrogenase. It leads to the formation of isovaleryl-CoA 3. Acyl-CoA dehydrogenase leading to the formation of beta-methylcrotonyl-CoA 4. Beta-methylcrotonyl-CoA carboxylase leads to the formation of beta-methylglutaconyl-CoA 5. The next step is catalyzed by the enzyme beta-methylglutaconyl-CoA hydratase 6. Final step is catalyzed by HMG-CoA lyase to yield acetyl-CoA and acetoacetate.

Figure 1.10.48. The pathway of tryptophan degradation. The enzymes involved are (1) tryptophan-2,3dioxygenase, (2) f ormamidase, (3) kynurenine-3-monooxygenase, (4) kynureninase (PLP dependent), (5) 3-hydroxyanthranilate- 3,4-dioxygenase, (6) amino carboxymuconate semialdehyde decarboxylase, (7) aminomuconate semialdehyde dehydrogenase, (8) hydratase, (9) dehydrogenase, and (10–16) enzymes of Reactions 5 through 11 in lysine degradation (Fig. 26-23). 2-Amino-3-carboxymuconate-6-semialdehyde, in addition to undergoing Reaction 6, spontaneously f orms quinolinate, an NAD and NADP precursor

The enzymes involved are: (Figure 1.10.48). 1. Tryptophan-2, 3-dioxygenase 2. Formamidase 3. Kynurenine-3-monooxygenase 4. Kynureninase (PLP dependent) 5. 3-hydroxyanthranilate 3,4-dioxygenase 6. Amino carboxymuconate semialdehyde decarboxylase 7. Aminomuconate semialdehyde dehydrogenase 8. Hydratase 9. Dehydrogenase 10. Alpha-keto acid dehydrogenase 11. Glutyrate-CoA dehydrogenase 12. Decarboxylase 13. Enoyl-CoA hydratase 14. Beta-hydoxyacyl-CoA dehydrogenase 15. HMG-CoA lyase

16. HMG-CoA lyase

Degradation of phenylalanine and Tyrosine to Fumarate and Acetoacetate

Phenylalanine hydroxylase (also called phenylalanine- 4-monooxygenase) is one of a general class of enzyme called mixed-function oxidases. all of which catalyze simultaneous hydroxylation of a substrate by an oxygen atom of O2 and reduction of the other oxygen atom to H2O. Phenylalanine hydroxylase, requires the cofactor tetrahydrobiopterin, which carries electrons from NADH to O2 and becomes oxidized to dihydrobiopterin in the process. It is subsequently reduced by the enzyme dihydrobiopterin reductase in a reaction that requires NADH. (Figure 1.10.49).

Figure 1.10.49. Catabolic pathways for phenylalanine and tyrosine. In humans these amino acids are normally converted to acetoacetyl-CoA and f umarate. Genetic def ects in many of these enzymes cause inheritable human diseases

Genetic disorders related to amino acid metabolism:

Alkaptonuria and Phenylketonuria Result from Defects in Phenylalanine Degradation-

 Archibald Garrod realized in the early 1900s that human genetic diseases result from specific enzyme deficiencies. The first such disease to be recognized was alkaptonuria, which, Garrod observed, resulted in the excretion of large quantities of homogentisic acid. This condition results from deficiency of homogentisate dioxygenase. Alkaptonurics suffer no ill effects other than arthritis later in life (although their urine darkens alarmingly because of the rapid air oxidation of the homogentisate they excrete.  Severe mental retardation occurs within a few months of birth if the disease is not detected and treated immediately. Indeed, approx.1% of the patients in mental institutions were, at one time (before routine screening), phenylketonurics.

Unit 1  PKU is caused by the inability to hydroxylate phenylalanine and therefore results in increased blood levels of phenylalanine (hyperphenylalaninemia). The excess phenylalanine is transaminated to phenylpyruvate, by an otherwise minor pathway.  The “spillover” of phenylpyruvate (a phenylketone) into the urine was the first observation connected with the disease and gave the disease its name, although it has since been demonstrated that it is the high concentration of phenylalanine itself that gives rise to brain dysfunction.  Classic PKU results from a deficiency in phenylalanine hydroxylase (PAH).It was the first human inborn error of metabolism whose basic biochemical defect had been identified. Because all of the tyrosine breakdown enzymes are normal, treatment consists in providing the patient with a lowphenylalanine diet and monitoring the blood level of phenylalanine to ensure that it remains within normal limits for the first 5 to 10 years of life (the adverse effects of hyperphenylalaninemia seem to disappear after that age).  PAH deficiency also accounts for another common symptom of PKU: Its victims have lighter hair and skin color than their siblings. This is because tyrosine hydroxylation, the first reaction in the formation of the black skin pigment melanin is inhibited by elevated phenylalanine levels.  These result from deficiencies in the enzymes catalyzing the formation or regeneration of 5, 6, 7, 8tetrahydrobiopterin (BH4), the PAH cofactor. In such cases, patients must also be supplied with L-

3,4-dihydroxyphenylalanine

 (L-DOPA) and 5-hydroxytryptophan, metabolic precursors of the neurotransmitters norepinephrine and serotonin, respectively, since tyrosine hydroxylase and tryptophan hydroxylase, the PAH homologs that produce these physiologically active amines, also require 5, 6, 7, 8-tetrahydrobiopterin

Defect in the lysine pathway:

 The saccharopine pathway is thought to predominate in mammals because a genetic defect in the enzyme that catalyzes  The first step in the sequence results in hyperlysinemia and hyperlysinuria (elevated levels of lysine in the blood and urine, respectively) along with mental and physical retardation. This is yet another example of how the study of rare inherited disorders has helped to trace metabolic pathway.

Maple Syrup Urine Disease Results from a Defect in Branched-Chain Amino Acid Degradation

 Branched-chain alpha-keto acid dehydrogenase (BCKDH; also known as alpha ketoisovalerate dehydrogenase), which  catalyzes second step of branched-chain amino acid degradation. A genetic deficiency in BCKDH causes maple syrup urine disease (MSUD), so named because the consequent buildup of branched-chain alpha-keto acids imparts the urine with the characteristic odor of maple syrup. Unless promptly treated by a diet low in branched-chain amino acids (but not too low because they are essential amino acids) MSUD is rapidly fatal. MSUD is an autosomal recessive disorder that is caused by defects in any of four of the complex’s six subunits, E1, E1, E2, or E3 (E1 is an alpha2beta2 heterotetramer).

Hyperhomocysteinemia Is Associated with Disease

 Imbalance between the rate of production of homocysteine through methylation reactions utilizing SAM and its rate of breakdown by either remethylation to form methionine or reaction with serine to form cystathionine in the cysteine biosynthesis pathway can result in an increase in the release of homocysteine to the extracellular medium and ultimately the plasma and urine. Moderately elevated concentrations of homocysteine in the plasma, hyperhomocysteinemia, for reasons that are poorly understood, are closely associated with cardiovascular disease, cognitive impairment, and neural