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.

Essential vs. Nonessential Amino Acids Nonessential Alanine Asparagine Aspartate Cysteine Glutamate Glutamine Glycine Proline Serine Tyrosine Essential Arginine* Histidine Isoleucine Leucine Lysine Methionine* Phenylalanine* Threonine Tyrptophan Valine

*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.

NON-ESSENTIAL AMINO ACID BIOSYNTHESIS

Glutamate and Aspartate Glutamate is synthesized from its' widely distributed -keto acid precursor by a simple 1-step transamination reaction catalyzed by glutamate dehydrogenase. As discussed in the Nitrogen Metabolism page, the glutamate dehydrogenase reaction plays a central role in overall nitrogen homeostasis.

Reactions of glutamate dehydrogenase 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.

Alanine and the Glucose-Alanine Cycle Aside from its role in protein synthesis, alanine is second only to glutamine in prominence as a circulating amino acid. In this capacity it serves a unique role in the transfer of nitrogen from peripheral tissue to the liver. Alanine is transferred to the circulation by many tissues, but mainly by muscle, in which alanine is formed from pyruvate at a rate proportional to intracellular pyruvate levels. Liver accumulates plasma alanine, reverses the transamination that occurs in muscle, and proportionately increases urea production. The pyruvate is either oxidized or converted to glucose via gluconeogenesis. When alanine transfer from muscle to liver is coupled with glucose transport from liver back to muscle, the process is known as the glucose-alanine cycle. The key feature of the cycle is that in 1 molecule, alanine, peripheral tissue exports pyruvate and ammonia (which are potentially rate-limiting for metabolism) to the liver, where the carbon skeleton is recycled and most nitrogen eliminated. There are 2 main pathways to production of muscle alanine: directly from protein degradation, and via the transamination of pyruvate by alanine transaminase, ALT (also referred to as serum glutamate-pyruvate transaminase, SGPT).

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 S-adenosylmethionine (SAM or AdoMet).

Biosynthesis of S-adenosylmethionine, SAM SAM serves as a precursor for numerous methyl transfer reactions (e.g. the conversion of norepinephrine to epinenephrine). The result of methyl transfer is the conversion of SAM to S-adenosylhomocysteine. Sadenosylhomocysteine is then cleaved by adenosylhomocyteinase to yield homocysteine and adenosine. Homocysteine can be converted back to methionine by methionine synthase, a reaction that occurs under methioninesparing conditions and requires N5-methyl-tetrahydrofolate as methyl donor. This reaction was discussed in the context of vitamin B12-requiring enzymes in the Vitamins page. Transmethylation reactions employing SAM are extremely important, but in this case the role of Sadenosylmethionine in transmethylation is secondary to the production of homocysteine (essentially a by-product of transmethylase activity). In the production of SAM all phosphates of an ATP are lost: one as P i and two as PPi. It is adenosine which is transferred to methionine and not AMP. In cysteine synthesis, homocysteine condenses with serine to produce cystathionine, which is subsequently cleaved by cystathionase to produce cysteine and -ketobutyrate. The sum of the latter two reactions is known as transsulfuration. Cysteine is used for protein synthesis and other body needs, while the -ketobutyrate is first converted to propionylCoA 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.

Utilization of Methionine in the Synthesis of Cysteine

The 2 key enzymes of this pathway, cystathionine synthase and cystathionase (cystathionine lyase), both use pyridoxal phosphate as a cofactor, and both are under regulatory control. Cystathionase is under negative allosteric control by cysteine, as well, cysteine inhibits the expression of the cystathionine synthase gene.
Genetic defects are known for both the synthase and the lyase. Missing or impaired cystathionine synthase leads to homocystinuria and is often associated with mental retardation, although the complete syndrome is multifaceted and many individuals with this disease are mentally normal. 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 pro-coagulant 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 B 12 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.

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 tetrahydrofolate-related cofactor tetrahydrobiopterin, which is maintained in the reduced state by the NADH-dependent enzyme dihydropteridine reductase (DHPR).

Biosynthesis of Tyrosine from Phenylalanine Missing or deficient phenylalanine hydroxylase results in hyperphenylalaninemia. Hyperphenylalaninemia is defined as a plasma phenylalanine concentration greater than 2mg/dL (120M). The most widely recognized hyperphenylalaninemia (and most severe) is the genetic disease known as phenlyketonuria (PKU). Patients suffering from PKU have plasma phenylalanine levels >1000M, whereas the non -PKU hyperphenylalaninemias exhibit levels of plasma phenylalanine <1000M. Untreated PKU leads to severe mental retardation. The mental re tardation is caused by the accumulation of phenylalanine, which becomes a major donor of amino groups in aminotransferase activity and depletes neural tissue of -ketoglutarate. This absence of -ketoglutarate in the brain shuts down the TCA cycle and the associated production of aerobic energy, which is essential to normal brain development.

The product of phenylalanine transamination, phenylpyruvic acid, is reduced to phenylacetate and phenyllactate, and all 3 compounds appear in the urine. The presence of phenylacetate in the urine imparts a "mousy" odor. If the problem is diagnosed early, the addition of tyrosine and restriction of phenylalanine from the diet can minimize the extent of mental retardation.
Because of the requirement for tetrahydrobiopterin in the function of phenylalanine hydroxylase, deficiencies in DHPR can manifest with hyperphenylalaninemia. However, since 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.

Ornithine and Proline Biosynthesis Glutamate is the precursor of both proline and ornithine, with glutamate semialdehyde being a branch point intermediate leading to one or the other of these 2 products. While ornithine is not one of the 20 amino acids used in protein synthesis, it plays a significant role as the acceptor of carbamoyl phosphate in the urea cycle. Ornithine serves an additional important role as the precursor for the synthesis of the polyamines. The production of ornithine from glutamate is important when dietary arginine, the other principal source of ornithine, is limited.

Synthesis of Ornithine and Proline from Glutamic Semialdehyde The fate of glutamate semialdehyde depends on prevailing cellular conditions. Ornithine production occurs from the semialdehyde via a simple glutamate-dependent transamination, producing ornithine. When arginine concentrations become elevated, the ornithine contributed 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.

Serine Biosynthesis The main pathway to de novo biosynthesis of serine starts with the glycolytic intermediate 3-phosphoglycerate. 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

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,N10-methylene-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. In addition, glycine functions in the central nervous system as an inhibitory neurotransmitter where it participates in regulating signals that process motor and sensory information that permit movement, vision and audition. Glycine is co-released with GABA which is the primary inhibitory neurotransmitter. Glycine action as a neurotransmitter is a function of the amino acid binding to a specific receptor, GlyR. GlyR is a member of the nicitinicoid receptor superfamily that includes the GABAA receptor (GABAAR), the excitatory nicotinic acetylcholine receptors (nAChR) and the serotonin type 3 receptor (5HT3). GlyR is a heteromeric complex consisting of a complex of either three or four -subunits and one -subunit. There are four different -subunit genes (1-4) and a single subunit gene in the human genome. GlyR is a ligand-gated ionotropic receptor that is a chloride channel. In addition to glycine, GlyR can be activated by several other small amino acids such as alanine and taurine. Glycine is also involved in the modulation of excitatory neurotransmission exerted via glutamate binding to N-methyl-D-aspartate (NMDA) receptors.

Aspartate/Asparagine and Glutamate/Glutamine Biosynthesis Glutamate is synthesized by the reductive amination of -ketoglutarate catalyzed by glutamate dehydrogenase; it is thus a nitrogen-fixing reaction. In addition, glutamate arises by aminotransferase reactions, with the amino nitrogen being donated by a number of different amino acids. Thus, glutamate is a general collector of amino nitrogen.

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.