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Essential and Nonessential Amino Acids Nonessential amino acids are those that are synthesized by mammals, while the essential amino acids must be obtained from dietary sources. Why would an organism evolve in such a way that it could not exist in the absence of certain amino acids? Most likely, the ready availability of these amino acids in lower organisms (plants and microorganisms) obviated the need for the higher organism to continue to produce them. The pathways for their synthesis were selected out. Not having to synthesize an additional ten amino acids (and regulate their synthesis) represents a major economy, then. Nevertheless, it remains for us to become familiar with the synthetic pathways for these essential amino acids in plants and microorganisms, and it turns out that they are generally more complicated that the pathways for nonessential amino acid synthesis and they are also species-specific. The twenty amino acids can be divided into two groups of 10 amino acids. Ten are essential and 10 are nonessential. However, this is really not an accurate dichotomy, as there is overlap between the two groups, as is indicated in the text accompanying the following two charts:
Introduction to Amino Acid Metabolism Essential versus Non-Essential Amino Acids Inborn Errors in Amino Acid Metabolism
Glutamine/Glutamate and Aspartate/Asparagine Alanine Arginine, Ornithine and Proline Glutamate and Aspartate Serine Alanine and the Glucose-Alanine Cycle Threonine Cysteine Glycine Tyrosine Cysteine Ornithine and Proline Methionine Serine Valine, Leucine, Isoleucine Glycine Phenylalanine and Tyrosine Aspartate/Asparagine and Glutamate/Glutamine Lysine Histidine Tryptophan
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Introduction
All tissues have some capability for synthesis of the non-essential amino acids, amino acid remodeling, and conversion of non-amino acid carbon skeletons into amino acids and other derivatives that contain nitrogen. However, the liver is the major site of nitrogen metabolism in the body. In times of dietary surplus, the potentially toxic nitrogen of amino acids is eliminated via transaminations, deamination, and urea formation; the carbon skeletons are generally conserved as carbohydrate, via gluconeogenesis, or as fatty acid via fatty acid synthesis pathways. In this respect amino acids fall into three categories: glucogenic, ketogenic, or glucogenic and ketogenic. Glucogenic amino acids are those that give rise to a net production of pyruvate or TCA cycle intermediates, such as -ketoglutarate or oxaloacetate, all of which are precursors to glucose via gluconeogenesis. All amino acids except lysine and leucine are at least partly glucogenic. Lysine and leucine are the only amino acids that are solely ketogenic, giving rise only to acetylCoA or acetoacetylCoA, neither of which can bring about net glucose production. A small group of amino acids comprised of isoleucine, phenylalanine, threonine, tryptophan, and tyrosine give rise to both glucose and fatty acid precursors and are thus characterized as being glucogenic and ketogenic. Finally, it should be recognized that amino acids have a third possible fate. During times of starvation the reduced carbon skeleton is used for energy production, with the result that it is oxidized to CO2 and H2O.
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*The amino acids arginine, methionine and phenylalanine are considered essential for reasons not directly related to lack of synthesis. Arginine is synthesized by mammalian cells but at a rate that is insufficient to meet the growth needs of the body and the majority that is synthesized is cleaved to form urea. Methionine is required in large amounts to produce cysteine if the latter amino acid is not adequately supplied in the diet. Similarly, phenyalanine is needed in large amounts to form tyrosine if the latter is not adequately supplied in the diet.
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Like glutamate, aspartate is synthesized by a simple 1-step transamination reaction catalyzed by aspartate aminotransferase, AST (formerly referred to as serum glutamate-oxalate transaminase, SGOT).
Aspartate can also be derived from asparagine (whose synthesis is outlined below) through the action of asparaginase. The importance of aspartate as a precursor of ornithine for the urea cycle is described in the Nitrogen Metabolism page.
The glucose-alanine cycle is used primarily as a mechanism for skeletal muscle to eliminate nitrogen while replenishing its energy supply. Glucose oxidation produces pyruvate which can undergo transamination to alanine. This reaction is catalyzed by alanine transaminase, ALT (ALT used to be called serum glutamate-pyruvate transaminase, SGPT). Additionally, during periods of fasting, skeletal muscle protein is degraded for the energy value of the amino acid carbons and alanine is a major amino acid in protein. The alanine then enters the blood stream and is transported to the liver. Within the liver alanine is converted back to pyruvate which is then a source of carbon atoms for gluconeogenesis. The newly formed glucose can then enter the blood for delivery back to the muscle. The amino group transported from the muscle to the liver in the form of alanine is converted to urea in the urea cycle and excreted.
Cysteine Biosynthesis
The sulfur for cysteine synthesis comes from the essential amino acid methionine. A condensation of ATP and methionine catalyzed by methionine adenosyltransferase yields Sadenosylmethionine (SAM or AdoMet).
Cysteine is used for protein synthesis and other body needs, while the -ketobutyrate is first converted to propionyl-CoA and then via a 3-step process to the TCA cycle intermediate succinyl-CoA. While cysteine readily oxidizes in air to form the disulfide cystine, cells contain little if any free cystine because the ubiquitous reducing agent, glutathione, effectively reverses the formation of cystine by a non-enzymatic reduction reaction.
Some instances of genetic homocystinuria respond favorably to pyridoxine therapy, suggesting that in these cases the defect in cystathionine synthase is a decreased affinity for the cofactor. Missing or impaired cystathionase leads to excretion of cystathionine in the urine but does not have any other untoward effects. Rare cases are known in which cystathionase is defective and operates at a low level. This genetic disease leads to methioninuria with no other consequences. Elevated levels of homocysteine in the blood have been shown to correlate with cardiovascular dysfunction. The role of homocysteine in cardiovascular disease is related to its ability to induce a state of inflammation. Homocysteine serves as a negatively charged surface that attracts the contact phase of the intrinsic pathway of blood coagulation. Activation of the intrinsic coagulation cascade leads to inappropriate thrombolytic events as well as resulting in increases in inflammatory cytokine release from leukocytes that are activated as a result of the procoagulant state. Therefore, it is important to ensure that proper function of the methionine synthase reaction is maintained. Although it would be assumed that increased intake of vitamin B12 should lead to increased conversion of homocysteine to methionine, and thus reduced levels of circulating homocysteine, controlled studies have shown that this does not occur.
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Tyrosine Biosynthesis
Tyrosine is produced in cells by hydroxylating the essential amino acid phenylalanine. This relationship is much like that between cysteine and methionine. Half of the phenylalanine required goes into the production of tyrosine; if the diet is rich in tyrosine itself, the requirements for phenylalanine are reduced by about 50%. Phenylalanine hydroxylase is a mixed-function oxygenase: one atom of oxygen is incorporated into water and the other into the hydroxyl of tyrosine. The reductant is the tetrahydrofolaterelated cofactor tetrahydrobiopterin, which is maintained in the reduced state by the NADHdependent enzyme dihydropteridine reductase (DHPR).
tetrahydrobiopterin is a cofactor in several other enzyme catalyzed reactions (e.g. see the synthesis of the tyrosine- and tryptophan-derived neurotransmitters as well as nitric oxide in Specialized Products of Amino Acids), the effects of missing or defective DHPR cause even more severe neurological difficulties than those usually associated with PKU caused by deficient phenylalanine hydroxylase activity.
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from the urea cycle plus that from glutamate semialdehyde inhibit the aminotransferase reaction, with accumulation of the semialdehyde as a result. The semialdehyde cyclizes spontaneously to 1-pyrroline-5-carboxylate which is then reduced to proline by an NADPH-dependent reductase.
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Serine Biosynthesis
The main pathway to de novo biosynthesis of serine starts with the glycolytic intermediate 3phosphoglycerate. An NADH-linked dehydrogenase converts 3-phosphoglycerate into a keto acid, 3-phosphopyruvate, suitable for subsequent transamination. Aminotransferase activity with glutamate as a donor produces 3-phosphoserine, which is converted to serine by phosphoserine phosphatase. As indicated below, serine can be derived from glycine (and visa versa) by a single step reaction that involves serine hydroxymethyltransferase and tetrahydrofolate (THF).
Serine Biosynthesis
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Glycine Biosynthesis
The main pathway to glycine is a 1-step reversible reaction catalyzed by serine hydroxymethyltransferase (SHMT). This enzyme is a member of the family of one-carbon transferases and is also known as glycine hydroxymethyltransferase. This reaction involves the
transfer of the hydroxymethyl group from serine to the cofactor tetrahydrofolate (THF), producing glycine and N5,N10-methylene-THF. There are mitochondrial and cytosolic versions of serine hydroxymethyltransferase. The cytosolic enzyme is referred to as SHMT1 and the mitochondrial enzyme is SHMT2.
Glycine produced from serine or from the diet can also be oxidized by glycine decarboxylase (also referred to as the glycine cleavage complex, GCC) to yield a second equivalent of N5,N10methylene-tetrahydrofolate as well as ammonia and CO2.
Glycine is involved in many anabolic reactions other than protein synthesis including the synthesis of purine nucleotides, heme, glutathione, creatine and serine.
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Aspartate is formed in a transamination reaction catalyzed by aspartate transaminase, AST. This reaction uses the aspartate -keto acid analog, oxaloacetate, and glutamate as the amino donor. Aspartate can also be formed by deamination of asparagine catalyzed by asparaginase.
Asparagine synthetase and glutamine synthetase, catalyze the production of asparagine and glutamine from their respective -amino acids. Glutamine is produced from glutamate by the
direct incorporation of ammonia; and this can be considered another nitrogen fixing reaction. Asparagine, however, is formed by an amidotransferase reaction.
Aminotransferase reactions are readily reversible. The direction of any individual transamination depends principally on the concentration ratio of reactants and products. By contrast, transamidation reactions, which are dependent on ATP, are considered irreversible. As a consequence, the degradation of asparagine and glutamine take place by a hydrolytic pathway rather than by a reversal of the pathway by which they were formed. As indicated above, asparagine can be degraded to aspartate.
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Asparaginase (see above) is also widely distributed within the body, where it converts asparagine into ammonia and aspartate. Aspartate transaminates to oxaloacetate, which follows the gluconeogenic pathway to glucose. Glutamate and aspartate are important in collecting and eliminating amino nitrogen via glutamine synthetase and the urea cycle, respectively. The catabolic path of the carbon skeletons involves simple 1-step aminotransferase reactions that directly produce net quantities of a TCA cycle intermediate. The glutamate dehydrogenase reaction operating in the direction of ketoglutarate production provides a second avenue leading from glutamate to gluconeogenesis.
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Alanine Catabolism
Alanine is also important in intertissue nitrogen transport as part of the glucose-alanine cycle (see above). Alanine's catabolic pathway involves a simple aminotransferase reaction that directly produces pyruvate. Generally pyruvate produced by this pathway will result in the formation of oxaloacetate, although when the energy charge of a cell is low the pyruvate will be oxidized to CO2 and H2O via the PDH complex and the TCA cycle. This makes alanine a glucogenic amino acid.
Serine Catabolism
The conversion of serine to glycine and then glycine oxidation to CO2 and NH3, with the production of two equivalents of N5,N10-methyleneTHF, was described above in the section on glycine biosynthesis. Serine can be catabolized back to the glycolytic intermediate, 3phosphoglycerate, by a pathway that is essentially a reversal of serine biosynthesis. However, the enzymes are different. Although it has been demonstrated in mammals such as rodents and dogs that serine can be converted to pyruvate through a deamination reaction catalyzed by serine/threonine dehydratase, this activity of the enzyme appears to be lacking in humans.
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Threonine Catabolism
There are at least 3 pathways for threonine catabolism that have been identified in yeasts, insects, and vertebrates including mammals. The principal threonine catabololizing pathway in humans involves glycine-independent serine/threonine dehydratase yielding -ketobutyrate which is further catabolized to propionyl-CoA and finally the TCA cycle intermediate, succinyl-CoA. Serine/threonine dehydratase is expressed at high levels only in the liver. It appears that in newborn infants catabolism of threonine occurs exclusively via the action of the serine/threonine dehydratase. Therefore, it is presumed that this is the predominant threonine catabolizing pathway in humans. The second pathway of threonine catabolism utilizes serine hydroxymethyltransferase. As indicated above this enzyme belongs to a family of one-carbon transferases and is alternatively named glycine hydroxymethyltransferase or threonine aldolase. The products of this reaction are acetyl-CoA and glycine. The glycine can be converted to serine via the same enzyme and the serine is then catabolized as described above yielding pyruvate and NH4+. Thus, via this catabolic pathway threonine yields ketogenic and glucogenic byproducts. In humans it appears that threonine aldolase is actually encoded by a non-functional pseudogene, whereas in other mammals and vertebrates (e.g. mice, zebrafish, and clawed frogs) the threonine aldolase gene encodes a functional threonine catabolizing enzyme. An additional pathway occurs in the mitochondria and is initiated by threonine dehydrogenase yielding -amino--ketobutyrate (2-amino-3-ketobutyrate). The 2-amino-3-ketobutyrate is either converted to acetyl-CoA and glycine, via the action of 2-amino-3-ketobutyrate coenzyme A ligase (also called glycine C-acetyltransfease), or it can spontaneously degrade to aminoacetone which is converted to pyruvate. The threonine dehydrogenase gene in humans appears to be nonfunctional due to the incorporation of three inactivating mutations. Thus, whereas, this enzyme is a major threonine catabolizing enzyme in other mammals such as mice, it is the serine/threonine dehydratase gene that is most important in threonine catabolism in humans.
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Glycine Catabolism
Glycine is classified as a glucogenic amino acid, since it can be converted to serine by serine hydroxymethyltransferase (see above), and serine can be converted back to the glycolytic intermediate, 3-phosphoglycerate or to pyruvate by serine/threonine dehydratase, although the latter reaction (serine to pyruvate) appears not be occur in human tissues. Nevertheless, the main glycine catabolic pathway leads to the production of CO2, ammonia, and one equivalent of N5,N10-methyleneTHF by the mitochondrial glycine decarboxylase, also called the glycine cleavage complex, GCC (see above).
Cysteine Catabolism
There are several pathways for cysteine catabolism. The simplest, but least important pathway is catalyzed by a liver desulfurase and produces hydrogen sulfide, (H2S) and pyruvate. The major catabolic pathway in animals is via cysteine dioxygenase that oxidizes the cysteine sulfhydryl to sulfinate, producing the intermediate cysteinesulfinate. Cysteinesulfinate can serve as a biosynthetic intermediate undergoing decarboxylation and oxidation to produce taurine. Catabolism of cysteinesulfinate proceeds through transamination to -sulfinylpyruvate which then undergoes desulfuration yielding bisulfite, (HSO3) and the glucogenic product, pyruvate. The enzyme sulfite oxidase uses O2 and H2O to convert HSO3 to sulfate, (SO4) and H2O2. The resultant sulfate is used as a precursor for the formation of 3'-phosphoadenosine-5'phosphosulfate, (PAPS). PAPS is used for the transfer of sulfate to biological molecules such as the sugars of the glycosphingolipids.
Other than protein, the most important product of cysteine metabolism is the bile salt precursor taurine, which is used to form the bile acid conjugates taurocholate and taurochenodeoxycholate.
The enzyme cystathionase can also transfer the sulfur from one cysteine to another generating thiocysteine and pyruvate. Transamination of cysteine yields -mercaptopyruvate which then reacts with sulfite, (SO32), to produce thiosulfate, (S2O32) and pyruvate. Both thiocysteine and thiosulfate can be used by the enzyme rhodanese to incorporate sulfur into cyanide, (CN), thereby detoxifying the cyanide to thiocyanate.
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Methionine Catabolism
The principal fates of the essential amino acid methionine are incorporation into polypeptide chains, and use in the production of -ketobutyrate and cysteine via SAM as described above. The transulfuration reactions that produce cysteine from homocysteine and serine also produce -ketobutyrate, the latter being converted first to propionyl-CoA and then via a 3-step process to succinyl-CoA. Regulation of the methionine metabolic pathway is based on the availability of methionine and cysteine. If both amino acids are present in adequate quantities, SAM accumulates and is a positive effector on cystathionine synthase, encouraging the production of cysteine and ketobutyrate (both of which are glucogenic). However, if methionine is scarce, SAM will form only in small quantities, thus limiting cystathionine synthase activity. Under these conditions accumulated homocysteine is remethylated to methionine, using N5-methylTHF and other compounds as methyl donors.
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The principal product from valine is propionylCoA, the glucogenic precursor of succinyl-CoA. Isoleucine catabolism terminates with production of acetylCoA and propionylCoA; thus isoleucine is both glucogenic and ketogenic. Leucine gives rise to acetylCoA and acetoacetylCoA, and is thus classified as strictly ketogenic. There are a number of genetic diseases associated with faulty catabolism of the BCAAs. The most common defect is in the branched-chain -keto acid dehydrogenase, BCKD. Since there is only one dehydrogenase enzyme for all three amino acids, all three -keto acids accumulate and are excreted in the urine. The disease is known as Maple syrup urine disease because of the characteristic odor of the urine in afflicted individuals. Mental retardation in these cases is extensive. Unfortunately, since these are essential amino acids, they cannot be heavily restricted in the diet; ultimately, the life of afflicted individuals is short and development is abnormal The main neurological problems are due to poor formation of myelin in the CNS.
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Lysine Catabolism
Lysine catabolism is unusual in the way that the -amino group is transferred to -ketoglutarate and into the general nitrogen pool. The reaction is a transamination in which the -amino group is transferred to the -keto carbon of -ketoglutarate forming the metabolite, saccharopine. Unlike the majority of transamination reactions, this one does not employ pyridoxal phosphate as a cofactor. Saccharopine is immediately hydrolyzed by the enzyme -aminoadipic semialdehyde synthase in such a way that the amino nitrogen remains with the -carbon of -ketoglutarate, producing glutamate and -aminoadipic semialdehyde. Because this transamination reaction is not reversible, lysine is an essential amino acid. The ultimate end-product of lysine catabolism is acetoacetyl-CoA. Genetic deficiencies in the enzyme -aminoadipic semialdehyde synthase have been observed in individuals who excrete large quantities of urinary lysine and some saccharopine. The lysinemia and associated lysinuria are benign. Other serious disorders associated with lysine metabolism are due to failure of the transport system for lysine and the other dibasic amino acids across the intestinal wall. Lysine is essential for protein synthesis; a deficiencies of its transport into the body can cause seriously diminished levels of protein synthesis. Probably more significant however, is the fact that arginine is transported on the same dibasic amino acid carrier, and resulting arginine deficiencies limit the quantity of ornithine available for the urea cycle. The result is severe hyperammonemia after a meal rich in protein. The addition of citrulline to the diet prevents the hyperammonemia. Lysine is also important as a precursor for the synthesis of carnitine, required for the transport of fatty acids into the mitochondria for oxidation. Free lysine does not serve as the precursor for this reaction, rather the modified lysine found in certain proteins. Some proteins modify lysine to trimethyllysine using SAM as the methyl donor to transfer methyl groups to the -amino of the lysine side chain. Hydrolysis of proteins containing trimethyllysine provides the substrate for the subsequent conversion to carnitine.
Histidine Catabolism
Histidine catabolism begins with release of the -amino group catalyzed by histidase, introducing a double bond into the molecule. As a result, the deaminated product, urocanate, is not the usual -keto acid associated with loss of -amino nitrogens. The end product of histidine catabolism is glutamate, making histidine one of the glucogenic amino acids. Another key feature of histidine catabolism is that it serves as a source of ring nitrogen to combine with tetrahydrofolate (THF), producing the 1-carbon THF intermediate known as N5formiminoTHF. The latter reaction is one of two routes to N5-formiminoTHF. Urocanate is converted to 4-imidazolone-5-propionate via the action of urocanate hydratase. The latter product is then converted to N-formiminoglutamte via the action of imidazolone propionase. Glutamate formiminotransferase then transfers the fomimino group to THF yielding glutamate and N5-formiminoTHF. The principal genetic deficiency associated with histidine metabolism is absence or deficiency of the first enzyme of the pathway, histidase. The resultant histidinemia is relatively benign. The disease, which is of relatively high incidence (1 in 10,000), is most easily detected by the absence of urocanate from skin and sweat, where it is normally found in relative abundance. Decarboxylation of histidine in the intestine by bacteria gives rise to histamine. Similarly, histamine arises in many tissues by the decarboxylation of histidine, which in excess causes constriction or dilation of various blood vessels. The general symptoms are those of asthma and various allergic reactions.
Synthesis of Histamine
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Tryptophan Catabolism
A number of important side reactions occur during the catabolism of tryptophan on the pathway to acetoacetate. The first enzyme of the catabolic pathway is an iron porphyrin oxygenase that opens the indole ring. The latter enzyme is highly inducible, its concentration rising almost 10fold on a diet high in tryptophan. Kynurenine is the first key branch point intermediate in the catabolic pathway leading to 3 fates: Kynurenine can undergo deamination in a standard transamination reaction yielding kynurenic acid. Kynurenic acid and metabolites have been shown to act as antiexcitotoxics and anticonvulsives. High levels of kynurenic acid have been found in the urine of individuals suffering from schizophrenia. Kynurenic acid has been shown to act as a non-competetive antagonist at the glycine binding site of the NMDA receptor (NMDA = N-methyl-D-aspartate) which is an ionotropic (ligand-gated ion channel) receptor for glutamate. The NMDA receptor is a key component of the glutaminergic neurotransmission system believed to be involved in the pathophysiology of schizophrenia, thus explaining the potential role of kynurenic acid in schizophrenia.
Kynurenine can also undergo a series of catabolic reactions producing 3-hydroxyanthranilic acid plus alanine. Another equivalent of alanine is produced from kynurenine in a single step reaction leading to anthranilic acid. It is the production of these alanine residues that allows tryptophan to be classified among the glucogenic amino acids. Oxidation of 3-hydroxyanthranilate converts it into 2-amino-3-carboxymuconic 6-semialdehyde, which has two fates. The main flow of carbon elements from this intermediate leads to acetoacetate which is why tryptophan is also a ketogenic amino acid. An important side reaction in liver involves a non-enzymatic cyclization to quinolate then via a transamination and several rearrangements yields limited amounts of nicotinic acid, which leads to production of a small amount of NAD+ and NADP+.
Aside from its role as an amino acid in protein biosynthesis, tryptophan also serves as a precursor for the synthesis of serotonin and melatonin. These products are discussed in Specialized Products of Amino Acids.
Note that tyrosine is really an essential amino acid, as it is synthesized by the hydroxylation of phenylalanine, an essential amino acid. Also, in animals, the sulfhydryl group of cysteine is derived from methionine, which is an essential amino acid, so cysteine can also be considered essential. The ten "essential" amino acids are:
Ignoring tyrosine (as it's immediate precursor is phenylalanine, an essential amino acid), all of the nonessential amino acids (and we will include arginine here) are synthesized from intermediates of major metabolic pathways. Furthermore, the carbon skeletons of these amino acids are traceable to their corresponding ketoacids. Therefore, it could be possible to synthesize any one of the nonessential amino acids directly by transaminating its corresponding
-ketoacid, if that ketoacid exists as a common intermediate. A "transamination reaction", in which an amino group is transferred from an amino acid to the -carbon of a ketoacid, is catalyzed by an aminotransferase. Three very common ketoacids can be transaminated in one step to their corresponding amino acid: Pyruvate (glycolytic end product) --> alanine Oxaloacetate (citric acid cycle intermediate) --> aspartate ketoglutarate (citric acid cycle intermediate) --> glutamate
Asparagine and glutamine are the products of amidations of aspartate and glutamate, respectively. Thus, asparagine and glutamine, and the remaining nonessential amino acids are not directly the result of transamination of -ketoacids because these are not common intermediates of the other pathways. Still, we will be able to trace the carbon skeletons of all of these back to an -ketoacid. I make this point not because of any profound implications inherent in it, but rather as a way to simplify the learning of synthetic pathways of the nonessential amino acids. Aspartate is transaminated to asparagine in an ATP-dependent reaction catalyzed by asparagine synthetase, and glutamine is the amino group donor:
The synthesis of glutamine is a two-step one in which glutamate is first "activated" to a glutamylphosphate intermediate, followed by a reaction in which NH3 displaces the phosphate group:
So, the synthesis of asparagine is intrinsically tied to that of glutamine, and it turns out that glutamine is the amino group donor in the formation of numerous biosynthetic products, as well as being a storage form of NH3 . Therefore, one would expect that glutamine synthetase, the enzyme responsible for the amidation of glutamate, plays a central role in the regulation of nitrogen metabolism. We will now look into this control in more detail, before proceeding to the biosynthesis of the remaining nonessential amino acids. You have previously studied the oxidative deamination of glutamate by glutamate dehydrogenase, in which NH3 and ketoglutarate are produced. The -ketoglutarate produced is then available for accepting amino groups in other transamination reactions, but the accumulation of ammonia as the other product of this reaction is a problem because, in high concentrations, it is toxic. To keep the level of NH3 in a controlled range, a rising level of ketoglutarate activates glutamine synthetase, increasing the production of glutamine, which donates its amino group in various other reactions.
The regulation of glutamine synthetase has been studied in E.Coli and, although complicated, it is worthwhile to look at some of its features because this will give us more insight into regulation of intersecting metabolic pathways. Xray diffraction of crystals of the enzyme reveals a
hexagonal prism structure (D6 symmetry) composed of 12 identical subunits. The activity of the enzyme is controlled by 9 allosteric feedback inhibitors, 6 of which are end products of pathways involving glutamine: histidine tryptophan carbamoyl phosphate (synthesized from carbamoyl phosphate synthetase II) glucosamine-6-phosphate AMP (see next lecture) CTP (see next lecture) The other three effectors are alanine, serine and glycine, which carry information regarding the cellular nitrogen level. The enzyme is also regulated by covalent modification (adenylylation of a Tyr residue), which results in an increase sensitivity to the cumulative feedback inhibition by the above nine effectors. Adenylyltransferase is the enzyme which catalyzes both the adenylylation and deadenylylation of E. coli glutamine synthetase, and this enzyme is complexed with a tetrameric regulatory protein, PII. Regulation of the adenylylation and its reverse occurs at the level of PII, depending upon the uridylylation of another Tyr residue, located on PII. When PII is uridylylated, glutamine synthetase is deadenylylated; the reverse occurs when UMP is covalently attached to the Tyr residue of PII. The level of uridylylation is, in turn, regulated by the activities of the two enzymes, uridylyltransferase and uridylyl-removing enzyme, both located on the same protein. Uridylyltransferase is activated by -ketoglutarate and ATP, while it is inhibited by glutamine and Pi. The following diagram summarizes the regulation of bacterial glutamine synthetase: see text pg 643
We can "walk through" this regulatory cascade by looking at a specific example, namely increased levels of -ketoglutarate ( reflecting a corresponding increase in NH3) levels: (1) Uridylyltransferase activity is increased (2) PII (in complex with adenylyltransferase) is uridylylated (3) Glutamine synthetase is deadenylylated
(4) -ketoglutarate and NH3 form glutamine and Pi That the control of bacterial glutamine synthetase is exquisitely sensitive to the level of the cell's nitrogen metabolites is illustrated by the fact that the glutamine just produced in the above cascade is now an inhibitor of further glutamine production.
Exercise: Use the regulatory pathway to explain the effect of a rising level of glutamine on the activity of bacterial glutamine synthetase.
Proline, Ornithine and Arginine are derived from Glutamate The first step involves phosphorylation of glutamate by ATP with the enzyme -glutamyl kinase, followed by reduction to glutamate-5-semialdehyde which spontaneously cyclizes (no enzyme required) to an internal Schiff base. The formation of the semialdehyde also requires the presence of either NADP or NADPH. The semialdehyde is a branch point, however. One branch leads to proline while the other branch leads to ornithine and arginine. Glutamate-5-semialdehyde is transaminated to ornithine and glutamate is the amino group donor. Ornithine, a urea cycle intermediate, is converted to arginine through the urea cycle.
To further highlight the importance of glutamate, it is converted to the physiologically active amine, -aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain:
Note the participation of glutamate as the amino group donor. Serine is converted to glycine in the following reaction: serine + THF --> glycine + N5,N10 -methylene-THF hydroxymethyltransferase) Glycine is also formed in a condensation reaction as follows: N5,N10 -methylene-THF + CO2 + NH4+ --> glycine requires NADH) (enzyme: glycine synthase; (enzyme: serine
ser + homocysteine -> cystathionine + H2O cystathionine + H2O --> -ketobutyrate + cysteine + NH3 Synthesis of Essential Amino Acids The synthetic pathways for the essential amino acids are: (1) present only in microorgansims (2) considerably more complex than for nonessential amino acids (3) use familiar metabolic precursors (4) show species variation For purposes of classification, consider the following 4 "families" which are based upon common precursors: (1) Aspartate Family: lysine, methionine,threonine (2) Pyruvate Family: leucine, isoleucine, valine (3) Aromatic Family: phenylalanine, Tyrosine, Tryptophan (4) Histidine
The first committed step for the synthesis of Lys, Met and Thr is the first step, in which aspartate is phosphorylated to aspartyl--phosphate, catalyzed by aspartokinase: E.coli has 3 isozymes of aspartokinase that respond differently to each of the 3 amino acids, with regard to enzyme inhibition and feedback inhibition. The biosynthesis of lysine, methionine and threonine are not, then, controlled as a group. The pathway from aspartate to lysine has 10 steps. The pathway from aspartate to threonine has 5 steps The pathway from aspartate to methionine has 7 steps Regulation of the three pathways also occurs at the two branch points: -Aspartate-semialdehyde (homoserine and lysine) Homoserine (threonine and methionine) The regulation results from feedback inhibition by the amino acid products of the branches, indicated in the brackets above.
We will consider one important step in the synthesis of this group of 3 amino acids, namely the step in which homocysteine is converted to methionine, catalyzed by the enzyme methionine synthase :
In this reaction, homocysteine is methylated to methionine, and the C1 donor is N5-methyl-THF. Note that the enzyme is called a "synthase" rather than a synthetase, because the reaction is a condensation reaction in which ATP (or another nucleoside triphosphate) is not used as an energy source. This is to be compared to a "synthetase" in which an NTP is required as an energy source.This reaction can also be looked at as the transfer of a methyl group from N5-methyl-THF to homocysteine, so another name for the enzyme catalyzing it is homocysteine methyltransferase. It is reasonable to review reactions in which a C1 unit is added to a metabolic precursor , as these reactions are seen very commonly in our study of biochemical pathways. You have already seen the transfer of a carboxyl group from the biotin cofactor of pyruvate carboxylase to pyruvate to form oxaloacetate (why isn't this called a "transferase" or a "synthase"?). Most carboxylation reactions use biotin as a cofactor. You have also studied methionine breakdown, in which the first step involves the transfer of adenosine to methionine to form SAdenosylmethionine (SAM). The methyl group on the sulfonium ion of SAM is highly reactive, so it is not surprising to find that SAM is a methylating agent in some reactions. Tetrahydrofolates are also C1 donating agents and, unlike the carboxylations and the SAM methylations, the THFs can transfer C1 units in more than one oxidation state. N5-methyl-THF ,as we have just seen, transfers the methyl group (-CH3), in which the oxidation level of C is that of methanol (-4). N5,N10-methylene-THF carries a methylene group (-CH2-) and the oxidation level is that of formaldehyde (0), while N5-formimino-THF transfers the formimino group (-CH=NH), in which the oxidation level of the C atom is that of formate. Formyl (-CH=O) and methenyl (-CH=) groups are also transfered by THF and these both have the C in the oxidation level of formate (+2). The structure of THF is suited for these transfers by virtue of its N5 and N10 groups as shown in the following chemical structure:
We will see THF again when we study the synthesis of thymidylate from dUMP, catalyzed by the enzyme thymidylate synthase in which N5,N10-methylene-THF is the methyl donor.
The Pyruvate Family These are the "branched chain" amino acids, and it's helpful to remember them as a group, not only because they all originate from the pyruvate carbon skeleton, but also because the disease "maple syrup urine disease" (MSUD) is a result of deficiency of branched-chain -ketoacid dehydrogenase, resulting in a buildup of branched-chain -keto acids. We'll just look at the beginning and the end of the pathways: The first step is common to all 3 amino acids: Pyruvate + TPP --> Hydroxyethyl-TPP (catalyzed by acetolactate synthase)
Note that the central carbon atom in hydroxyethyl-TPP is a carbanion and it is stabilized by resonance forms. Hydroxyethyl-TPP can react with another pyruvate to form -acetolactate, in which case the pathway heads toward valine and isoleucine, or it can react with -ketobutyrate, in which case the pathway leads to isoleucine.
There is a branch point at -ketoisovalerate which, in one direction leads to valine and, in the other, to leucine. The final step in the formation of each of these amino acids involves the transfer of an amino group from glutamate to the corresponding -ketoacid of each of the 3 branched-chain amino acids.Here we see another example of the importance of one particular amino acid, namely glutamate, to the anabolic pathways for amino acids. The Aromatic Amino Acids: Phosphoenolpyruvate (PEP), a glycolytic intermediate, condenses with erythrose-4-phosphate, a pentose-phosphate pathway intermediate, to form 2-keto-3-deoxyarabinoheptulosonate-7phosphate and inorganic phosphate. The enzyme involved is a synthase. This condensation product eventually cyclizes to chorismate.
From here, the pathway branches, ending up in the production of tryptophan at one branch end, and tyrosine and phenylalanine at the other end. A few high points deserve mention. First, glutamine plays a role as the donor of an amino group to chorismate to form anthranilate at the tryptophan branch.The immediate precursor of tryptophan is indole:
The "indole ring" is the characterizing feature of the tryptophan structure. Note that serine is the donor of the amino group to indole to form tryptophan. The branch that leads towards tyrosine and phenylalanine has another branch point at prephenate. The only difference between the 2 resulting amino acids is that the para carbon of the benzene ring of tyrosine is hydroxylated. Indeed, in mammals, phenylalanine is directly hydroxylated to tyrosine, catalyzed by the enzyme phenylalanine hydroxylase. Clinical Correlation: Defective or absent phenylalanine hydroxylase results in buildup of phenylalanine, which is subsequently transaminated to phenylpyruvate and excreted in the urine. The disease "phenylketonuria" rapidly leads to severe mental retardation if not treated soon after birth with a low phenylalanine diet. Universal screening of newborns in the U.S. for this condition by blood analysis has greatly decreased the morbidity of the untreated condition.
Some very important physiologically active amines are derived from tyrosine, and these are LDOPA, dopamine, norepinephrine and epinephrine. The pathway from tyrosine to norepinephrine is shown below:
The formation of epinephrine from norepinephrine involves the transfer of the highly reactive methyl group of S-adenosylmethionine to norepinephrine:
Histidine Biosynthesis: We will look at this pathway in a bit more detail, because it involves the molecule 5phosphoribosyl--pyrophosphate (which we will refer to as "PRPP" from now on). PRPP is also involved in the synthesis of purines and pyrimidines, as we will soon see. In the first step of histidine synthesis, PRPP condenses with ATP to form a purine, N1-5'-phosphoribosyl ATP, in a reaction that is driven by the subsequent hydrolysis of the pyrophosphate that condenses out. Glutamine again plays a role as an amino group donor, this time resulting in the formation of 5aminoamidazole-4-carboximide ribonucleotide (ACAIR), which is an intermediate in purine biosynthesis. Histidine is special in that its biosynthesis is inherently linked to the pathways of nucleotide formation. Histidine residues are often found in enzyme active sites, where the chemistry of the imidazole ring of histidine makes it a nucleophile and a good acid/base catalyzer. We now know that RNA can have catalytic properties, and there has been speculation that life was originally RNA-based. Perhaps the transition to protein catalysis from RNA catalysis occurred at the origin of histidine biosynthesis.
In the next lecture, we will look at the synthesis of nucleotides. Since we have just identified histidine as a likely evolutionary transition point from purines to amino acids and proteins, this is a convenient point at which to transition from one lecture to another.