Amino Acid Metabolism

This page describes the amino acids that are important in human nutrition. It covers the digestion and absorbtion of proteins in the gut, some of the amino acid degradation pathways in the tissues, and the urea cycle. There are separate pages dealing with nucleotide and 'one carbon' metabolism and with porphyrin metabolism. Other aspects of amino acid biochemistry are covered in the section on the integration of metabolism. Click here to skip over the detailed contents section.

Protein turnover

The recommended minimal protein intake required to achieve nitrogen balance in healthy adults is about 50g per day, although in developed countries many people may eat double this amount. This compares with an average daily protein turnover of about 250g per day. Human proteins have very different lifetimes. Total body protein is about 11kg, but about 25% of this is collagen, which is metabolically inert. A typical muscle protein might survive for three weeks, but many liver enzymes turn over in a couple of days. Some regulatory enzymes have half-lives measured in hours or minutes. The majority of the amino acids released during protein degradation are promptly re-incorporated into fresh proteins. Net protein synthesis accounts for less than one third of the dietary amino acid intake, even in rapidly growing children consuming a minimal diet. Most of the ingested protein is ultimately oxidised to provide energy, and the surplus nitrogen is excreted, a little as ammonia but mostly as urea.

Soluble intracellular proteins are tagged for destruction by attaching ubiquitin, a low molecular weight protein marker. They are then degraded in proteasomes to short peptides. A very few of these are displayed on the cell surface by the MHC [major histocompatibility] complex as part of the immune system, but most of them are further metabolised to free amino acids. Some proteins are degraded by an alternative system within the lysosomes.

Dietary proteins are initially denatured by the stomach acid, in conjunction with limited proteolysis by pepsin. In young mammals gastric rennin [do not confuse with renin!] partially hydrolyses and precipitates milk casein and increases gastric residence time. Gastric acid also kills most ingested bacteria, rendering the upper part of the gut almost sterile.

Protein digestion is largely completed in the small intestine at a slighlty alkaline pH. The pancreatic proteases trypsin, chymotrypsin and elastase divide the proteins into short peptides. These are attacked from both ends by aminopeptidase and carboxypeptidase, and the fragments are finished off by dipeptidases secreted from the gut wall.

Amino acid uptake from the gut lumen into enterocytes is driven by the sodium gradient. There is a relatively high sodium concentration in the gut (regardless of dietary intake, as a result of the pancreatic secretion of sodium bicarbonate) and a low concentration in the enterocytes, as a result of the sodium pump in the basolateral membrane. A multiplicity of sodium-linked amino acid carriers operate within the intestinal brush border, balanced by sodium-independent export carriers on the serosal surface (i.e. the opposite side) of the cells.

Central role of glutamate

Four of the amino acids: glutamate, aspartate, alanine and glutamine are present in cells at much higher concentrations than the other 16. All four have major metabolic functions in addition to their roles in proteins, but glutamate occupies the prime position.

Glutamate and aspartate function as excitatory neurotransmitters in the central nervous system, and glutamate is partly responsible for the flavour of food. (It is the mono sodium glutamate listed on processed food labels.) However, glutamate also occupies a special position in amino acid breakdown, and most of the nitrogen from dietary protein is ultimately excreted from the body via the glutamate pool.

Glutamate is special because it is chemically related to 2-oxoglutarate (= alpha keto glutatarate) which is a key intermediate in the citric acid (Krebs) cycle. Glutamate can be reversibly converted into oxoglutarate by transaminases or by glutamate dehydrogenase. In addition, glutamate can be reversibly converted into glutamine, an important nitrogen carrier, and the most common free amino acid in human blood plasma.

Transamination reactions

Most common amino acids can be converted into the corresponding keto acid by transamination. This reaction swops the amino group from one amino acid to a different keto acid, thereby generating a new pairing of amino acid and keto acid. There is no overall loss or gain of nitrogen from the system - it is simply a question of "robbing Peter to pay Paul".

Transamination reactions are readily reversible, and the equilibrium constant is close to 1. One of the two pairs is almost invariably glutamate and its corresponding keto acid oxoglutarate, although there are a few exceptions to this rule. All transaminases require pyridoxal phosphate (derived from vitamin b6) as a cofactor.

The substrates bind to the active centre one at a time, and the function of the pyridoxal phosphate is to act as a temporary store of amino groups until the next substrate comes along. In the process the pyridoxal phosphate is converted into pyridoxamine phosphate, and then back again. Enzymologists call this a "ping pong" mechanism, and it leads to a characteristic pattern in the reaction kinetics.

The condensation between the alpha amino group and the aromatic aldehyde to form a "Schiff base" makes the alpha carbon atom chemically reactive, so the isomerisation of the Schiff base takes place very easily. In practice the pyridoxal form of the coenzyme condenses with the epsilon amino group of a lysine residue in the enzyme protein when no amino acid is bound, and the free aldehyde form of the coenzyme has only a transitory existence. Many of the enyzmes that metabolise amino acids require pyridoxal phosphate as a cofactor. Unexpectedly, this compound also serves in a different manner in the active centre of glycogen phosphorylase.

Glutamate:oxaloacetate transaminase [GOT]

This enzyme is also known as aspartate aminotransferase and is one of the most active enzymes in the cell. It exists in mitochondrial and cytosolic variants, and the detailed iso-enzyme pattern is tissue-specific. It escapes in large amounts from dead or dying tissues and enters the bloodstream, so GOT is often measured in blood samples for medical diagnostic purposes.

The metabolic importance of this enzyme is that it brings about a free exchange of amino groups between glutamate (which is the most common amino acid) and aspartate which is a second major amino acid pool. Glutamate and aspartate are each required for separate but essential steps in the urea cycle, which is responsible for ammonia detoxication and nitrogen excretion. The free movement of nitrogen between the glutamate and aspartate pools is an important balancing process that is vital for normal cellular metabolism.

This reaction is close to equilibrium in both the cytosol and the mitochondrial compartments. It forms an integral part of the malate - asparate shuttle which is effectively responsible for the "transport" of NADH across the inner mitochondrial membrane.

Glutamate:pyruvate transaminase [GPT]

This very active enzyme is also known as alanine aminotransferase and exists in mitochondrial and cytosolic variants. The detailed iso-enzyme pattern is tissue-specific. It escapes in large amounts from dead or dying tissues and GPT may be measured in blood samples for medical diagnostic purposes.

Alanine is the principal amino acid released from muscle tissue during starvation. It is an important substrate for hepatic gluconeogenesis, and alanine transamination is required for the proper maintenance of fasting blood glucose concentrations.

Glutamate dehydrogenase [GluDH]

This enzyme is the first committed step on the final common pathway for mammalian nitrogen excretion, leading eventually to urea. A few of the amino acids have specific deamination pathways, but about 75% of ingested protein nitrogen follows the glutamate route.

Glutamate dehydrogenase in mammals is almost entirely confined to the liver mitochondrial matrix space, where it accounts for a significant proportion of the total protein. In contrast to the transamination reactions which merely swop amino groups from one compound to another, GluDH catalyses a net loss of nitrogen from the amino acid pool. The process is therefore termed "oxidative deamination". It is the only common dehydrogenase which is non-specific for NAD or NADP, and this may be important for its overall regulation.

NADH / NAD and NADPH / NADP have the same standard redox potential of -420mV when the oxidised and reduced forms are present in equal concentrations. In practice these coenzymes have different effective redox potentials and perform specialised functions within cells. The NADPH / NADP pool operates almost entirely in the reduced form, but the NADH / NAD pool is rarely more than 30% reduced. In general NADPH is used to drive reductive biosynthetic reactions, whereas NAD is the coenzyme for the oxidative energy-yielding pathways.

The dual coenzyme specificity is a potential source of difficulty for the cell, since in theory this readily reversible enzyme could catalyse a futile cycle, proceeding first in the oxidative direction with NAD, followed by a reductive step using NADPH. The effect would be to "short circuit" the two coenzyme pools, which normally require considerable investment in substrates and cellular equipment to keep them separate. If this futile cycle happens to any significant extent then it must be an advantage for the cell, because it has persisted unchanged for 2,000,000,000 years of evolutionary development.

The most likely explanation at present is that this futile cycle takes place, but for various reasons it does not place an excessive burden on its owner. The Km of GluDH for ammonia is quite high, and the free ammonia concentration is kept very low by the next enzyme in the pathway, carbamyl phosphate synthetase. This will severely reduce the rate of the synthetic reaction, and allow the enzyme to catalyse a net glutamate oxidation at a slow controlled rate that provides the maximum opportunity for regulatory interference.

Regulation is plainly critical at this point, since GluDH and carbamyl phosphate synthetase jointly control the overall rate of nitrogen excretion and determine whether a particular individual will be in positive, neutral or negative nitrogen balance. Control of unwanted nitrogen losses remains an important unsolved problem after major surgery, burns or other serious traumatic injuries.

The enzyme is indeed modulated by adenine and guanine nucleotides, although it is difficult to make much sense of the observed effects. GluDH has all the hallmarks of a large multimeric allosteric enzyme, although the true nature of the regulation

Remains to be identified. The situation is in some ways similar to the parallel NAD and NADP linked oxidation pathways for malate and isocitrate, although the competing reactions for these substrates are separately regulated and catalysed by different proteins.
The liver GluDH gene is located on chromosome 10. OMIM link Surp

risingly few genetic defects have been reported, possibly reflecting the catastrophic nature of the resulting fault. There are reports of an additional GluDH gene located on the X chromosome OMIM link which is expressed in testis and neural tissues. This seems to be a processed pseudogene that is still functional.

Trans-deamination

Most transaminases share a common substrate and product (glutamate and oxoglutarate) with glutamate dehydrogenase, and this permits a combined nitrogen excretion pathway for individual amino acids that is commonly described as "trans-deamination".

This process underlines the central role of glutamate in the overall control of nitrogen metabolism.

Urea cycle

Ammonium ions are in equilibrium with about 1% free ammonia at physiological pH. Ammonium salts are toxic compounds, causing vomiting, convulsions and ultimately coma and death when the blood concentration exceeds approximately 0.25mM. It is not entirely clear why this should be so: it may be that ammonium ions mimic potassium ions, but gain access as uncharged ammonia to areas from which they should be excluded. Alternatively, they may favour the synthesis of excessive amounts of glutamate and glutamine which have excitatory effects on neural tissues.

It is therefore necessary to have an efficient means to remove ammonia from the body. Water-living species commonly excrete free ammonia through their gills [ammonotelism], but this easy option is not available to land dwellers which produce a variety of less toxic nitrogenous end products. Urea synthesis and excretion [ureotelism] first evolved in lungfish and primitive amphibia about 400 million years ago. The process is replicated today when ammonotelic tadpoles leave the water and metamorphose into ureotelic frogs. Urea is also used in humans, and in all placental mammals, which start to express the urea cycle genes around the time of birth. Urea is very soluble, but still requires appreciable quantities of water for its removal via the kidneys. This imposes a minimum daily water requirement and limits the range of environments that these species can exploit.

Urea is not the only possible solution to the problem: spiders excrete guanine, which packs no less than 5 surplus nitrogen atoms into a single small molecule, while reptiles and birds excrete mainly uric acid [uricotelism]. Uric acid is an extremely insoluble purine compound that readily forms supersaturated solutions. This has been turned to advantage in uricotelic species, which can survive in extremely arid environments. They regurgitate concentrated urine, supersaturated with uric acid, from the cloaca into the hindgut where the uric acid crystalises and the residual water is resorbed. The uric acid forms the fine pasty mass of white crystals that is familiar to us in bird droppings.

Uricotelism is also an advantage to animals that lay shelled eggs, which of necessity have a zero water intake. The uric acid crystalises within the allantois, part of which eventually becomes incorporated into the lower gut as the embryo develops. In humans the insolubility of uric acid is a considerable nuisance, since it gives rise to the extremely painful deposits of small crystals [called "tophi"] within the joints of patients suffering from gout.

Urea is synthesised via the urea cycle, which is confined to mammalian liver. Individual enzymes from the urea cycle are present in other tissues, and may be important for arginine biosynthesis, but the complete cycle does not occur. Extra-hepatic tissues export their surplus nitrogen to the liver by other routes, principally as the amino acids alanine and glutamine. In addition, the cleavage of arginine by nitric oxide synthetase generates citrulline, which is a urea cycle intermediate. Citrulline is recycled to arginine, and in tissues which use the nitric oxide signalling system the relevant urea cycle enzymes have sufficient activity to maintain cellular arginine supplies.

The urea cycle takes place partly in the cytosol and partly in the mitochondria, and the individual reactions are as follows.

carbamyl phosphate synthetase 1 [CPS1]

This mitochondrial enzyme converts the ammonia produced by glutamate dehydrogenase into carbamyl phosphate (=carbamoyl phosphate) which is an unstable high energy compound. It is the mixed acid anhydride of carbamic acid and phosphoric acid, and requires two molecules of ATP to drive its synthesis.

CPS1 is strongly activated by N-acetyl glutamate, which controls the overall rate of urea production. This bizarre method of regulation is not fully understood: N-acetyl glutamate is an intermediate in the bacterial synthesis of ornithine, but this feature has been lost from mammals and only the regulatory system has survived. There is a futile cycle catalysed by the enzymes N-acetylglutamate synthetase and N-acetylglutamate hydrolase. This is plainly important for the control of nitrogen metabolism, but we do not yet know how it works.

CPS1 deficiency results in hyperammonemia. OMIM link The neonatal cases are usually lethal, but there is also a less severe, delayed-onset form. Ammonia-dependent CPS1 is present only in the liver mitochondrial matrix space. It should be distinguished from a second cytosolic glutamine-dependent carbamyl phosphate synthetase [CPS2] which is found in all tissues and is involved in pyrimidine biosynthesis. Carbamyl phosphate synthesis is a major burden for liver mitochondria. This enzyme accounts for about 20% of the total protein in the matrix space. Glutamate dehydrogenase is also present in very large amounts.

ornithine transcarbamylase [OTCase]

The next reaction also takes place in the liver mitochondrial matrix space, where ornithine is converted into citrulline.

This enzyme has no regulatory significance. However, the OTCase gene is on the X chromosome and an inherited deficiency is observed in males, with an incidence of about 1 in 80,000 people. OMIM link This is the most common of the inherited urea cycle defects. Patients show all the symptoms of hyperammonemia, and in addition may excrete abnormal amounts of orotate, since the unused carbamyl phosphate escapes into the cytosol and enters the pyrimidine biosynthetic pathway.

ornithine and citrulline porters

The remainder of the urea cycle takes place in the cytosol. This requires the continuous export of citrulline and the uptake of ornithine across the inner mitochondrial membrane. These processes are catalysed by specific amino acid porters, which are present only in liver mitochondria. A very rare deficiency state has been described.

Glutamate and glutamate:aspartate porters

Urea production requires continuous mitochondrial glutamate uptake, to replenish the substrate for the glutamate dehydrogenase reaction. This process is catalysed by a specific electroneutral glutamate / hydroxyl antiporter, which is largely confined to liver mitochondria.

In addition, depending on the diet, mitochondria may also need to export aspartate in exchange for glutamate in order to balance the supplies of nitrogen to the mitochondrial and cytosolic segments of the urea cycle. This electrical process is driven by the mitochondrial membrane potential and is discussed more fully in connection with the malate - aspartate cycle.

arginino-succinate synthetase

Once in the cytosol, citrulline condenses with aspartate and the reaction is driven by ATP. In this way aspartate contributes the second nitrogen atom to urea, the first having come from glutamate.

Production of arginino-succinate is an energetically expensive process, since the ATP is split to AMP and pyrophosphate. The pyrophosphate is then cleaved to inorganic phosphate using pyrophosphatase, so the overall reaction costs two equivalents of high energy phosphate per mole.

arginino-succinate lyase

Elimination of fumarate from arginino-succinate then yields arginine.

This reaction sequence is very similar to the conversion of IMP to AMP in the purine biosynthetic pathway. In each case fumarate is formed as a by-product. Fumarate is not transported by mitochondria, so this requires the presence of cytosolic fumarase to form malate.

fumarate + H₂O = malate

The reaction is readily reversible, and the equilibrium slightly favours malate. The cytosolic and mitochondrial fumarase isoenzymes are extremely similar and derived from the same gene through alternative mRNA splicing reactions. OMIM link

Cleavage of arginine by arginase to produce urea regenerates ornithine, which is then available for another round of the cycle.

The transcriptional regulation of the urea cycle genes was reviewed by Takaguchi & Mori (1995) Biochem. J. 312, 649 - 659. Unfortunately this document is not available on line, but a more recent wide-ranging review on arginine metabolism (Wu & Morris (1998) Biochem. J. 336, 1 - 17) and an article by Kimura et al (1998) J. Biol. Chem. 273, 27505 - 27510 can both be downloaded in Adobe portable document format. In essence, the urea cycle enzymes appear around the time of birth, and thereafter reflect the rate of amino acid catabolism, either from dietary sources or from tissue breakdown. The principal inducing stimuli are glucagon and glucocorticoids, and the main repressor is insulin. The CCAAT/enhancer binding protein (C/EBP) seems to play a major role. Urea cycle regulation and control of blood ammonia levels are impaired in C/EBP knockout mice.

Nitric Oxide

In addition to its metabolic functions in the urea cycle, arginine is also the immediate precursor for nitric oxide [NO], an important signalling molecule involved in the local regulation of blood flow. Nitric oxide synthase uses oxygen and NADPH, and the other products are citrulline and NADP.

The citrulline is reconverted to arginine using two of the urea cycle enzymes, although the full urea cycle does not take place outside the liver. See also the review by Wu & Morris mentioned above.

Essential and non-essential amino acids

Humans can degrade all the amino acids that are commonly found in proteins, but we have very limited synthetic capacity. However, the initial transamination step in most of the amino acid breakdown pathways is freely reversible. If the corresponding keto acids are produced during normal metabolism, then it may be possible to use surplus nitrogen from other sources to make good a dietary deficiency in some of these "non-essential" amino acids, providing that the total nitrogen intake is sufficient.

In addition, a few amino acids are degraded to form other amino acids (for example, phenylalanine is metabolised initially to tyrosine) so that tyrosine is essential on a minimal diet, but becomes non-essential if sufficient phenylalanine is eaten. Tyrosine is therefore described as a "conditionally essential" amino acid.

Relatively few keto acids and amino acids can be produced from alternative sources, so about half of the amino acids are essential in the diet. [See table below.]

Glycogenic and ketogenic amino acids

The carbon skeletons from the majority of amino acids are degraded to Krebs cycle intermediates after removal of the amino group by transamination. This means that they can give rise to blood glucose via the gluconeogenic pathway. They are termed 'glycogenic' amino acids, because it was observed many years ago that they made diabetic glycosuria worse. In contrast to this 'ketogenic' amino acids exacerbated diabetic ketoacidosis, and these amino acids are degraded to compounds such as acetoacetate and acetyl-CoA. 'Mixed' amino acids are degraded to both Krebs cycle acids and to acetyl-CoA.

Glutamine metabolism

In view of the toxicity of free ammonia and ammonium salts, cells require a non-toxic source of nitrogen for use in nitrogen transport and biosynthetic reactions. This need is satisfied by glutamine, which is the most common free amino acid in human blood plasma.

Glutamine is readily synthesised from glutamate and ammonium ions by the enzyme glutamine synthetase. This enzyme is present in liver and in many other body tissues. It has a low Km for ammonium, and works efficiently at non-toxic ammonium concentrations. The required energy comes from ATP:

Glutamine supplies most of the nitrogen required for purine and pyrimidine biosynthesis, and for the manufacture of amino sugars. When necessary it can be degraded back to glutamate by the enzyme glutaminase:

glutamine + H₂O = glutamate + NH₄⁺

Glutaminase is activated by inorganic phosphate. It is obvious that glutamine synthetase and glutaminase constitute a potential futile cycle, and the arrangment must be delicately regulated to avoid wasteful hydrolysis of ATP. The two enzymes are commonly present in different cells, and this seems to be particularly important in the liver and in the central nervous system. In liver, the urea cycle enzymes, glutamate:pyruvate transaminase and glutaminase are concentrated in the periportal cells, whereas glutamine synthetase is concentrated in the perivenous cells near the hepatic veins. See, for example, Racine et al (1995) Biochem. J. 305, 263-268; Dingemanse et al (1996) Hepatology 24, 407-411 and also Lie-Venema et al (1997) Biochem. J. 323, 611-619. Subscribers can download the last article here in Adobe pdf format.

In addition to the numerous biosynthetic uses for glutamine, kidney tubules can use ammonia derived from glutamine to control the urinary pH, and avoid large cation losses under acidotic conditions. In neural tissues, formation of glutamine is thought to terminate the action of glutamate, an excitatory neurotransmitter. Glutamine is a particularly important fuel for the cells lining the gut, and has been used experimentally in the treatment of gastrointestinal disease. Large amounts of glutamine and alanine are released from muscle cells during starvation, and these are important substrates for hepatic gluconeogenesis under fasting conditions.

Phenylketonuria

Phenylalanine is normally metabolised by conversion to tyrosine. The enzyme responsible for this conversion is phenylalanine hydroxylase, a mixed function oxygenase with a tetrahydrobiopterin cofactor:

Half of the oxygen molecule re-appears in the tyrosine -OH group and the other half is reduced to water. The "dihydrobiopterin" in the above reaction is an isomer of the folic acid compounds involved in one-carbon metabolism. It is recycled back to tetrahydrobiopterin using NADH:

"dihydrobiopterin" + NADH = tetrahydrobiopterin + NAD

Approximately one person in 45 American whites is a carrier for a defective phenylalanine hydroxylase gene, or (less frequently) the dihydrobiopterin cofactor. These mutations are particularly common in people of Celtic origin, but are less frequent in Eastern Europe. OMIM link Blood phenylalanine levels are elevated, but otherwise these heterozygotes show no symptoms and may even enjoy a heterozygote advantage. Homozygotes occur about 1 in 8,000 live births (45 * 45 * 4) and are very severely affected. Unless treated they are seriously mentally defective and excrete large quantities of phenylpyruvate in the urine. This compound gives the disease its name, and is formed by the transamination of phenylalanine, a reaction that is normally insignificant. These patients are often tyrosine deficient, and have abnormally light skin pigmentation, because they have insufficient tyrosine to synthesise melanin in normal amounts.

When this condition was first recognised in the 1930's, a significant proportion of all long term patients in mental institutions proved to be undiagnosed phenylketonuriacs. Nowadays the condition can be readily diagnosed by a heel-prick blood test performed on all new-born babies.

Treatment consists of a very low phenylalanine diet, supplemented with extra tyrosine that the patients cannot synthesise from phenylalanine. This diet must be instituted at birth, but can be discontinued after a few years when brain maturation is completed. For obvious reasons it must be restarted during pregnancy. The artificial sweetner aspartame is a phenylalanine derivative, and this fact is declared on soft drinks cans to assist those following the special diet.

Methyl malonic aciduria

Four amino acids: isoleucine, methionine, threonine and valine are degraded to propionyl CoA. Some additional propionate is formed in the gut from bacterial fermentation, and a little from the oxidation rare odd-numbered fatty acids. Patients with a metabolic block in the vitamin B12 dependent conversion of propionyl CoA to succinyl CoA suffer from a severe metabolic acidosis, and excrete large quantities of methyl malonic acid in their urine.