Source: BiochemFFA_6_5.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
In contrast to some of the metabolic pathways described to this point, amino acid metabolism is not a single pathway. The 20 amino acids have some parts of their metabolism that overlap with each other, but others are very different from the rest. In discussing amino acid metabolism, we will group metabolic pathways according to common metabolic features they possess (where possible). First, we shall consider the anabolic pathways.
Before beginning discussion of the pathways, it is worthwhile to discuss a reaction common to the metabolism of most of the amino acids and other nitrogen-containing compounds and that is transamination. In cells, nitrogen is a nutrient that moves from one molecule to another in a sort of hand-off process. A common transamination reaction is shown on the next page.
A specific reaction of this type is shown in Figure 6.134.
Glutamate and glutamine play central roles in transamination, each containing one more amine group than α-ketoglutarate and glutamate, respectively. Transamination reactions, as noted earlier, occur by a ping-pong mechanism and involve swaps of amines and oxygens in Schiff base reactions. Two amino acids, glutamine and asparagine are the products of gaining an amine in their respective R-groups in reactions involving ammonium ion.
It is also important to recognize that organisms differ considerably in the amino acids that they can synthesize. Humans, for example, cannot make 9 of the 20 amino acids needed to make proteins, and the number of these that can be synthesized in needed amounts varies between adults and children.
Amino acids that cannot be made by an organism must be in the diet and are called essential amino acids. Non-essential amino acids are those an organism can make in sufficient quantities (Figure 6.135). Though amino acids do not have a common pathway of metabolism, they are often organized in “families” of amino acids with overlapping metabolic reactions common to members of each group. To designate amino acid families in the text we will use a blue font for headings to distinguish them.
This family of amino acids arises from α-ketoglutarate of the citric acid cycle. It includes the amino acids glutamic acid, glutamine, proline, and arginine. It is also called the glutamate family, since all the amino acids in it derive from glutamate.
α-ketoglutarate is readily converted to glutamate in transamination reactions, as noted above. It can also be produced by the enzyme glutamate dehydrogenase, which catalyzes the reaction below (in reverse) to make glutamate.
In the forward direction, the reaction is a source of ammonium ion, which is important both for the urea cycle and for glutamine metabolism. Because it is a byproduct of a citric acid cycle intermediate, glutamate can therefore trace its roots to any of the intermediates of the cycle. Citrate and isocitrate, for example, can be thought of as precursors of glutamate. In addition, glutamate can be made by transamination from α-ketoglutarate in numerous transamination reactions involving other amino acids.
Synthesis of glutamine proceeds from glutamate via catalysis of the enzyme glutamine synthetase, one of the most important regulatory enzymes in all of amino acid metabolism (Figure 6.136).
Regulation of the enzyme is complex, with many allosteric effectors. It can also be controlled by covalent modification by adenylylation of a tyrosine residue in the enzyme (Figure 6.137). In the figure, PA and PD are regulatory proteins facilitating conversion of the enzyme.
Ammonia used in the reaction catalyzed by glutamate synthetase commonly arises from nitrite reduction, amino acid breakdown, or photorespiration. Because it builds ammonia into an amino acid, glutamine synthetase helps reduce the concentration of toxic ammonia - an important consideration in brain tissue. Some inhibitors of glutamine synthetase are, in fact, the products of glutamine metabolism. They include histidine, tryptophan, carbamoyl phosphate, glucosamine-6-phosphate, CTP, and AMP. The glutamate substrate site is a target for the inhibitors alanine, glycine, and serine. The ATP substrate site is a target for the inhibitors GDP, AMP, and ADP. Complete inhibition of the enzyme is observed when all of the substrate sites of the multi-subunit enzyme are bound by inhibitors. Lower levels of inhibitors results in partial or full activity, depending on the actual amounts.
Synthesis of proline starts with several reactions acting on glutamate. They are shown below in the green text box.
The L-glutamate-5-semialdehyde, so produced, is a branch point for synthesis of proline or ornithine. In the path to make proline, spontaneous cyclization results in formation of 1-pyrroline-5-carboxylic acid (Figure 6.138).
This, in turn, is reduced to form proline by pyrroline-5-carboxylate reductase.
Arginine is a molecule synthesized in the urea cycle and, thus, all urea cycle molecules can be considered as precursors. Starting with citrulline, synthesis of arginine can proceed as shown on the next page. The urea cycle can be seen HERE.
An alternate biosynthetic pathway for making arginine from citrulline involves reversing the reaction catalyzed by nitric oxide synthase. It catalyzes an unusual five electron reduction reaction that proceeds in the following manner
Yet another way to synthesize arginine biologically is by reversal of the arginase reaction of the urea cycle
Arginine can also be made starting with glutamate. This 5 step pathway leading to ornithing is illustrated at the top of the next page (enzymes in blue). Ornithine, as noted above can readily be converted to arginine.
The last means of making arginine is by reversing the methylation of asymmetric dimethylarginine (ADMA - Figure 6.140). ADMA is a metabolic byproduct of protein modification. It interferes with production of nitric oxide and may play a role in cardiovascular disease, diabetes mellitus, erectile dysfunction, and kidney disease.
Serine is a non-essential amino acid synthesized from several sources. One starting point is the glycolysis intermediate, 3-phosphoglycerate, (3-PG) in a reaction catalyzed by 3-PG dehydrogenase.
Transamination by phosphoserine aminotransferase produces O-phosphoserine. The phosphate is then removed by phosphoserine phosphatase, to make serine. These reactions are shown below. Phosphoserine phosphatase is missing in the genetic disease known as Williams-Beuren syndrome.
Serine can also be derived from glycine and vice versa. Their metabolic paths are intertwined as will be seen below. Serine is important for metabolism of purines and pyrimidines, and is the precursor for glycine, cysteine, and tryptophan in bacteria, as well as for sphingolipids and folate. Serine in the active site of serine proteases is essential for catalysis. A serine in the active site of acetylcholinesterases is the target of nerve gases and insecticides.
Covalent modification target
Serine in proteins can be the target of glycosylation or phosphorylation. D-serine is the second D-amino acid known to function in humans. It serves as a neuromodulator for NMDA receptors, by serving as a co-agonist, together with glutamate. D-serine is being studied as a schizophrenia treatment in rodents and as a possible biomarker for Alzheimers.
As noted, glycine’s metabolism is intertwined with that of serine. This is apparent in the reaction catalyzed by serine hydroxymethyltransferase.
Notably, the previous reaction is also needed for recycling of folate molecules, which are important for single carbon reactions in nucleotide synthesis.
Vertebrates can also synthesize glycine in their livers using the enzyme glycine synthase.
Glycine is a very abundant component of collagen. It is used in the synthesis of purine nucleotides and porphyrins. It is an inhibitory neurotransmitter and is a co-agonist of NMDA receptors with glutamate. Glycine was detected in material from Comet Wild 2.
Cysteine can be synthesized from several sources. One source is the metabolism of the other sulfur-containing amino acid, methionine. This begins with formation of S-Adenosyl-Methionine (SAM), catalyzed by methionine adenosyltransferase.
SAM is a methyl donor for methyl transfer reactions and that is the next step in the pathway - donation of a methyl group (catalyzed by transmethylase)
SAH (S-Adenosylhomocysteine) is cleaved by S-adenosylhomocysteine hydrolase,
Homocysteine can be recycled back to methionine by action of methionine synthase
On the path to making cysteine, homocysteine reacts as follows (catalyzed by cystathionine β-synthase).
Last, cystathionase catalyzes release of cysteine
β-ketobutyrate can be metabolized to propionyl-CoA and then to succinyl-CoA to be used ultimately in the citric acid cycle.
Another route to making cysteine is a two-step process that begins with serine, catalyzed first by serine-O-acetyltransferase
and then by cysteine synthase
Cysteine can be also released from cystine by cystine reductase
Finally, cysteine can be made from cysteic acid by action of cysteine lyase
Metabolism of aspartic acid is similar to that of glutamate. Aspartic acid can arise from transamination of a citric acid cycle intermediate (oxaloacetate).
Aspartate can also be generated from asparagine by the enzyme asparaginase.
Further, aspartate can be produced by reversal of a reaction in the urea cycle (see HERE)
Aspartate is also a precursor to four amino acids that are essential in humans. They are methionine, isoleucine, threonine, and lysine. Because oxaloacetate can be produced from aspartate, aspartate is an important intermediate for gluconeogenesis when proteins are the energy source.
Asparagine, too, is an amino acid produced in a simple transamination reaction. In this case, the precursor is aspartate and the amine donor is glutamine (catalyzed by asparagine synthetase)
Metabolism of methionine overlaps with metabolism of the other sulfur-containing amino acid, cysteine. Methionine is not made in humans (essential) so the pathway shown in Figure 6.141 is from bacteria.
The process begins with phosphorylation of aspartate. Numbers for each catalytic step in the figure are for the enzymes that follow:
1 - Aspartokinase
2 - Aspartate-semialdehyde dehydrogenase
3 - Homoserine dehydrogenase
4 - Homoserine O-transsuccinylase
5 - Cystathionine-γ-synthase
6 - Cystathionine-β-lyase
7 - Methionine synthase
Though humans cannot make methionine by the pathway shown in the figure, they can recycle methionine from homocysteine (a product of S-adenosylmethionine metabolism). This reaction requires the enzyme methionine synthase and Vitamin B12 as a co-factor.
An alternative pathway of converting homocysteine to methionine involves a prominent liver enzyme, betaine-homocysteine methyltransferase. This enzyme catalyzes the reaction below.
In this reaction, a methyl group is transferred to homocysteine from glycine betaine to make the methionine. Glycine betaine is a trimethylated amine of glycine found in plants. It is a byproduct of choline metabolism.
Bacteria, mitochondria, and chloroplasts use a modified form of methionine, N-formyl-methionine (Figure 6.142), as the first amino acid incorporated into their proteins. Formylation of methionine occurs only after methionine has been attached to its tRNA for translation. Addition of the formyl group is catalyzed by the enzyme methionyl-tRNA formyltransferase
Though threonine is chemically similar to serine, the metabolic pathway leading to threonine does not overlap with that of serine. As seen in the figure, aspartate is a starting point for synthesis. Two phosphorylations/dephosphorylations and two reductions with electrons from NADPH result in production of threonine.
Enzymes in Figure 6.143 are as follows:
2 β-aspartate semialdehyde dehydrogenase
3 Homoserine dehydrogenase
4 Homoserine kinase
5 Threonine synthase
Breakdown of threonine produces acetyl-CoA and glycine. It can also produce α-ketobutyrate, which can be converted to succinyl-CoA for oxidation in the citric acid cycle.
To get from aspartate to lysine, nine reactions and two non-enzymatic steps are involved, as seen in Figure 6.144. Enzymes involved in lysine biosynthesis include (numbers correspond to numbered reactions in Figure 6.144):
1 - Aspartokinase
2 - Aspartate-semialdehyde dehydrogenase
3 - 4-hydroxy-tetrahydrodipicolinate synthase
4 - 4-hydroxy-tetrahydrodipicolinate reductase
5 - 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase
6 - Succinyl-diaminopimelate transaminase
7 - Succinyl-diaminopimelate desuccinylase
8 - Diaminopimelate epimerase
9 - Diaminopimelate decarboxylase
Low in cereal grains
Lysine is the essential amino acid found in the smallest quantity in cereal grains, but is found abundantly in legumes. Besides its synthesis and breakdown, lysine can be methylated, acetylated, hydroxylated, ubiquitinated, sumoylated, neddylated, biotinylated, pupylated, and carboxylated within proteins containing it. Hydroxylation of lysine is important for strengthening collagen and acetylation/methylation of lysine in histone proteins play roles in control of gene expression and epigenetics. Besides being used to make proteins, lysine is important for calcium absorption, recovery from injuries, and for production of hormones.
Oral lysine has been used as a treatment for herpes infections (cold sores) but its efficacy is not established and it is not clear by what mechanism is would reduce the duration of the infection or reduce the number of outbreaks of viral infection..
Aromatic amino acids
The aromatic amino acids, tryptophan, phenylalanine, and tyrosine can all be made starting with two simple molecules - PEP and erythrose-4-phosphate (Figure 6.145). All three aromatic amino acids are also important sources of hormones, neurotransmitters, and even the skin pigment melanin.
The proteogenic amino acid with the largest R-group, tryptophan is an essential amino acid distinguished structurally by its indole group. The amino acid is made in bacteria and plants from shikimic acid or anthranilate and serine is used in its synthesis.
Erythrose-4-phosphate and phosphenolpyruvate (PEP) also serve as building blocks of tryptophan. The pathway of its synthesis is shown in Figures 6.146 to 6.148.
Erythrose-4-phosphate and phosphoenolpyruvate (PEP) are joined and then, after one hydrolysis, one dehydration, one oxidation and one reduction, the product is shikimic acid (Figure 6.147).
Shikimic acid is converted to chorismic acid in three steps, as shown in Figure 6.147. Finally, synthesis of tryptophan from chorismic acid is shown in Figure 6.148.
Regulation of tryptophan synthesis in bacteria occurs partly via a process called attenuation that operates through the trp operon. In this mechanism, low levels of tryptophan slow ribosomal movement (and translation) through the operon. This is particularly important because bacteria can have transcription and translation occurring simultaneously. Slowing translation due to low tryptophan levels allows a transcription termination mechanism to be inhibited. Since translation only slows when tryptophan is in short supply, premature termination of transcription occurs when tryptophan is abundant (see also HERE).
Besides its importance for making proteins, tryptophan is an important precursor of serotonin (neurotransmitter), melatonin (hormone), niacin (vitamin), and auxin (plant hormone). The two pathways leading from tryptophan to three of these molecules is shown in Figure 6.149.
Melatonin is a compound made from tryptophan that is found in a wide spectrum of biological systems, including plants, animals, fungi, and bacteria. In animals, it acts as a hormone for circadian rhythm synchronization, signaling the onset of darkness each day. It has effects on the timing of sleep, seasonal effects, and can affect blood pressure, among other physiological phenomena. It can cross cell membranes, as well as the blood-brain barrier. Melatonin is a potent anti-oxidant and provides protective functions for nucleic acids. It is used sometimes to help in treatment of sleep disorders. Some reports have indicated that children with autism have abnormal melatonin pathways with low levels of the hormone.
Melatonin production is affected by blue light and may be linked to sleep abnormalities for people using computer monitors after dark. To protect against this, some computer programs are available that reduce the screen’s blue light output in the evenings. Special eyeglasses that block blue light are also available. Though melatonin is linked to sleep in some animals (including humans), nocturnal animals are activated by increasing melatonin levels. Varying day/night lengths during the year alter melatonin production and provide biological signals of the seasons. These are especially important in the seasonal coloring and breeding habits of some animals. Melatonin is present in cherries, bananas, grapes, rice, cereals, olive oil, wine, and beer.
Serotonin, or 5-hydroxytryptamine, is a monoamine neurotransmitter derived from tryptophan. Blood platelets store serotonin and release it when they bind to a clot, causing vasoconstriction. Serotonin plays a role in cognitive functions and enhances memory and learning. Serotonin is widely thought to be a contributor to feelings of happiness and well-being. Some common anti-depressant drugs, including Prozac, Paxil and Zoloft, act to modulate action of serotonin at synapses.
Niacin is also known as Vitamin B3 and nicotinic acid. Niacin can be made from tryptophan and people who have the inability to absorb tryptophan in the digestive system exhibit symptoms similar to niacin deficiency.
Extreme deficiency of niacin in the diet leads to the disease known as pellagra, while insufficient amounts of niacin in the diet are linked with nausea, anemia, headaches, and tiredness. A diet that is primarily composed of grains like corn can lead to niacin deficiency, because the niacin in these sources is not readily bioavailable. Treatment of the grain with alkali, as in the traditional Mexican practice of soaking corn in lime, can make the niacin more easily absorbed from food.
Niacin is related to pyridine and the amide form of it is nicotinamide, an important component of NAD+/NADH and NADP+/NADPH. The last pairs of molecules are essential as electron acceptors/carriers for most cellular oxidation-reduction reactions.
Auxins are plant growth hormones derived from tryptophan. The most important of these is indole-3-acetic acid (Figure 6.151). Auxins are involved in almost every aspect of plant growth and development. They activate proteins, such as expansins and various enzymes that modify the structure of cell wall components, to loosen the cell walls of a plant and stimulate elongation of cells. In the presence of cytokinins, auxins stimulate cell division. Auxins are also involved in the maintenance of meristems and in cell patterning and organogenesis. Auxins are crucial for establishing root primordia as well as for elongation of root hairs. Auxins play important roles in organizing the xylem and phloem of plants, and it has long been known that plant callus tissue can be made to differentiate into shoots or roots, depending on the relative concentrations of auxins and cytokinins supplied in the medium.
Agrobacterium tumefaciens, a bacterium which infects a wide variety of plants, inserts its own DNA, including genes necessary for the synthesis of plant hormones, into its host’s cells. The subsequent overproduction of auxins stimulates the growth of tumors (called crown galls) on the plant (Figure 6.153).
Phenylalanine is an essential, hydrophobic amino acid in humans that is a precursor of tyrosine and since tyrosine is a precursor of several important catecholamines, phenylalanine is, thus, a precursor of them as well.
Phenylalanine is linked to the genetic disease phenylketonuria (PKU) which arises from an inability to metabolize the amino acid in people lacking (or deficient in) the enzyme phenylalanine hydroxylase. If left untreated, the disease can cause brain damage and even death, but if detected early, it can be easily managed by carefully monitoring dietary intake of the amino acid. Because of this, newborns are routinely tested for PKU. Phenylalanine is a component of the artificial sweetener known as aspartame (Nutrasweet - Figure 6.154) and is consequently dangerous for people suffering from this disorder.
Biosynthesis of phenylalanine in bacteria overlaps with synthesis of tryptophan. The branch occurs at chorismic acid where the enzyme chorismate mutase catalyzes a molecular rearrangement to produce prephenate.
Proton attack on prephenate results in loss of water and carbon dioxide to yield phenylpyruvate.
Transamination of phenylpyruvate yields phenylalanine.
Alternatively, phenylalanine can obtain its amine group in a transamination reaction from alanine.
Hydroxylation of phenylalanine by aromatic amino acid hydroxylase (phenylalanine hydroxylase) yields tyrosine.
Because tyrosine is made from phenylalanine and the latter is an essential amino acid in humans, it is not clear whether to classify tyrosine as essential or non-essential. Some define it as a conditionally essential amino acid. Others simply categorize it as non-essential.
As noted above, tyrosine can arise as a result of hydroxylation of phenylalanine. In addition, plants can synthesize tyrosine by oxidation of prephenate followed by transamination of the resulting 4-hydroxyphenylpyruvate (Figure 6.155).
The hydroxyl group on tyrosine is a target for phosphorylation by protein kinase enzymes involved in signal transduction pathways (Figure 6.156). When located in membranes, these enzymes are referred to as receptor tyrosine kinases and they play important roles in controlling cellular behavior/response.
In photosystem II of chloroplasts, tyrosine, at the heart of the system, acts as an electron donor to reduce oxidized chlorophyll. The hydrogen from the hydroxyl group of tyrosine is lost in the process, requiring re-reduction by four core manganese clusters.
Tyrosine is also important in the small subunit of class I ribonucleotide reductases where it forms a stable radical in the catalytic action of the enzyme (see HERE).
Tyrosine is a precursor of catecholamines, such as L-dopa, dopamine, norepinephrine, and epinephrine (Figure 6.157). The thyroid hormones triiodothyronine (T3) and thyroxine (T4) are also synthesized from tyrosine. As shown in Figure 6.158, this involves a series of iodinations of tyrosines side-chains of a protein known as thyroglobulin. Combinations of iodinated tyrosines give rise to thyroxine and triiodothyronine. These are subsequently cleaved from the protein and released into the bloodstream.
Oxidation and polymerization of tyrosine is involved in synthesis of the family of melanin pigments. Tyrosine is involved in the synthesis of at least two types - eumelanin and pheomelanin (Figure 6.159).
Another molecule derived from tyrosine is the benzoquinone portion of Coenzyme Q (CoQ). This pathway requires the enzyme HMG-CoA Reductase and since this enzyme is inhibited by cholesterol-lowering statin drugs, CoQ can be limited in people being treated for high cholesterol levels.
Dopamine plays several important roles in the brain and body. A member of the catecholamine and phenethylamine families, its name comes from the fact that it is an amine made by removing a carboxyl group from L-DOPA. Dopamine is synthesized in the brain and kidneys. It is also made in plants, though its function in plants is not clear. Conversion of dopamine to norepinephrine (Figure 6.157) requires vitamin C.
Dopamine is a neurotransmitter, being released by one nerve cell and then traveling across a synapse to signal an adjacent nerve cell. Dopamine plays a major role in the brain’s reward-mediated behavior. Rewards, such as food or social interaction, increase dopamine levels in the brain, as do addictive drugs. Other brain dopamine pathways are involved in motor control and in managing the release of various hormones.
Outside the nervous system, dopamine is a local chemical messenger. In blood vessels, it inhibits norepinephrine release and causes vasodilation. In the kidneys, it increases sodium excretion and urine output. It reduces gastrointestinal motility and protects intestinal mucosa in the digestive system and in the immune system, it reduces lymphocyte activity. The effect dopamine has on the pancreas is to reduce insulin production. With the exception of the blood vessels, dopamine is synthesized locally and exerts its effects near the cells that release it.
Epinephrine (also called adrenalin) is a catecholamine chemically related to norepinephrine that is a hormone with medical applications. It is used to treat anaphylaxis, cardiac arrest, croup, and, in some cases, asthma, when other treatments are not working, due to its ability to favor bronchodilation.
Epinephrine is the drug of choice for treating anaphylaxis. The compound may be given through inhalation, by intravenous injection, or subcutaneous injection and exerts effects through the α- and β-adrenergic receptors. In the body, it is produced and released by adrenal glands and some neurons.
Physiological effects of epinephrine may include rapid heart beat, increased blood pressure, heart output, pupil dilation, blood sugar concentration and increased sweating. Other physical effects may include shakiness, increased anxiety, and an abnormal heart rhythm.
Norepinephrine (also called noradrenalin) is a catecholamine molecule that acts as a hormone and neurotransmitter. It is chemically similar to epinephrine, differing only in the absence of a methyl group on its amine. Norepinephrine is made and released by the central nervous system (locus coeruleus of the brain) and the sympathetic nervous system. The compound is released into the blood stream from adrenal glands and affects α- and β-adrenergic receptors.
Norepinephrine is at its lowest levels during sleep and at its highest levels during stress (fight or flight response). The primary function of norepinephrine is to prepare the body for action. It increases alertness, enhances memory functions, and helps to focus attention. Norepinephrine increases heart rate and blood pressure, increases blood glucose and blood flow to skeletal muscle and decreases flow of blood to the gastrointestinal system.
Norepinephrine may be injected to overcome critically low blood pressure and drugs countering its effects are used to treat heart conditions. α-blockers, for example, are used to battle cardiovascular and psychiatric disorders. β-blockers counter a different set of norepinephrine’s effects than α-blockers and are used to treat glaucoma, migraine headaches and other cardiovascular problems.
The family of amino acids derived from pyruvate has four members, each with a simple aliphatic side chain no longer than four carbons. The simplest of these is alanine.
Alanine is the amino acid that is most easily produced from pyruvate. The simple transamination catalyzed by alanine transaminase produces alanine from pyruvate.
Alternative pathways for synthesis of alanine include catabolism of valine, leucine, and isoleucine.
The glucose-alanine cycle is an important nitrogen cycle related to the Cori cycle that occurs between muscle and liver cells in the body (see HERE). In it, breakdown of glucose in muscles leads to pyruvate. When nitrogen levels are high, pyruvate is transaminated to alanine, which is exported to hepatocytes.
In the liver cells, the last transamination of the glucose-alanine cycle occurs. The amine group of alanine is transferred to α-ketoglutarate to produce pyruvate and glutamate. Glucose can then be made by gluconeogenesis from pyruvate. Importantly, breakdown of glutamate yields ammonium ion, which can be made into urea for excretion, thus reducing the body’s load of potentially toxic amines. This pathway may be particularly important in the brain.
Another way of removing excess ammonium from a tissue is by attaching it to glutamate to make glutamine. Glutamate is a neurotransmitter, so having an alternative way of removing amines (glucose-alanine cycle) is important, especially in the brain.
Like valine and isoleucine, leucine is an essential amino acid in humans. In adipose tissue and muscle, leucine is used in sterol synthesis. It is the only amino acid to stimulate muscle protein synthesis, and as a dietary supplement in aged rats, it slows muscle degradation. Leucine is an activator of mTOR, a protein which, when inhibited, has been shown to increase life span in Saccharomyces cerevisiae, C. elegans, and Drosophila melanogaster.
Metabolism of leucine, valine, and isoleucine (also called Branched Chain Amino Acids - BCAAs) starts with decarboxylation of pyruvate and attachment of the two-carbon hydroxyethyl fragment to thiamine pyrophosphate (Figure 6.161). Metabolism of isoleucine proceeds with attachment of the hydroxylated two carbon piece (hydroxyethyl-TPP) to α-ketobutyrate and is covered in the section describing that amino acid (see HERE).
Metabolism of valine and leucine proceeds with attachment of the hydroxyethyl piece from TPP to another pyruvate to create α-acetolactate. Rearrangement of α-acetolactate by acetolactate mutase makes 3-hydroxy-3-methyl-2-oxobutanoate.
Reduction with NAD(P)H by acetohydroxy acid isomeroreductase yields α,β-dihydroxyisovalerate.
Loss of water, catalyzed by dihydroxyacid dehydratase produces α-ketoisovalerate.
This molecule is a branch point for synthesis of leucine and valine. Addition of an acetyl group from acetyl-CoA yields α-isopropylmalate (catalyzed by α-isopropylmalate synthase).
Rearrangement, catalyzed by isopropylmalate dehydratase, gives rise to β-isopropylmalate.
Oxidation by isopropylmalate dehydrogenase and NAD+, gives α-ketoisocaproate.
Transamination of it (catalyzed by leucine aminotransferase and using glutamate) gives the final product of leucine (top of next column).
An essential amino acid in humans, valine is derived in plants from pyruvate and shares part of its metabolic synthesis pathway with leucine and a small slice of it with isoleucine. Metabolism of all three amino acids starts with decarboxylation of pyruvate and attachment of the two-carbon hydroxyethyl fragment to thiamine pyrophosphate (Figure 6.161), as noted above.
As seen earlier, α-ketoisovalerate is the molecule at the point in the metabolic pathway where synthesis of valine branches from that of leucine. In fact, α-ketoisovalerate is only one step away from valine. Transamination of α-ketoisovalerate catalyzed by valine isoleucine aminotransferase gives valine.
Synthesis of isoeleucine (an essential amino acid in humans) begins in plants and microorganisms with pyruvate and α-ketobutyrate (a byproduct of threonine metabolism - threonine deaminase - Figure 6.162).
Metabolism of isoleucine proceeds with attachment to α-ketobutyrate of the hydroxyethyl-TPP product of pyruvate decarboxylation to form α-aceto-α-hydroxybutyrate. The reaction is catalyzed by acetolactate synthase. Rearrangement and reduction by acetohydroxy acid isomeroreductase and NAD(P)H yields α,β-dihydroxy-β-methylvalerate. Shown on next page.
Loss of water (catalyzed by dihydroxy acid dehydratase) gives α-keto-β-methylvalerate.
Τransamination (using glutamate and valine isoleucine transaminase) yields isoleucine.
Interestingly, several of the enzymes of valine metabolism catalyze reactions in the isoleucine pathway. Though the substrates are slightly different, they are enough like the valine intermediates that they are recognized as substrates.
Isoleucine has a second asymmetric center within it, but only one isomeric form of the four possible ones from the two centers is found biologically.
Regulation of synthesis
Regulation of synthesis of the branched chain amino acids (BCAAs - valine, leucine, and isoleucine) is complex. The key molecule in the regulation is α-ketobutyrate, which is synthesized in cells as a breakdown product of threonine. The enzyme catalyzing its synthesis is threonine deaminase (Figure 6.162), which is allosterically regulated. The enzyme is inhibited by its own product (isoleucine) and activated by valine, a product of a parallel pathway.
Thus, when valine concentration is high, the balances shifts in favor of production of isoleucine and since isoleucine competes with valine and leucine for hydroxyethyl-TPP, synthesis of these two amino acids goes down. When isoleucine concentration increases, threonine deaminase is inhibited, shifting the balance back to production of valine and leucine.
Another control mechanism for regulation of leucine synthesis occurs in bacteria and is known as attenuation. In this method, accumulation of leucine speeds the process of translation of a portion of the mRNA copy of the leucine operon (coding sequences for enzymes necessary to make leucine). This, in turn, causes transcription of the genes of the leucine operon to terminate prematurely, thus stopping production of the enzymes necessary to make leucine.
When leucine levels fall, translation slows, preventing transcription from terminating prematurely and allowing leucine metabolic enzymes to be made. Thus, leucine levels in the cell control the synthesis of enzymes necessary to make it.
Synthesis of histidine literally occurs in a class by itself - there are no other amino acids in its synthesis family. The amino acid is made in plants (Arabidopsis, in this case) by a pathway that begins with ribose-5-phosphate. The overall pathway is show in the green text boxes on the next two pages. Abbreviations used in the boxes are shown below.
1 = Ribose-phosphate diphosphokinase
3 = Phosphoribosyl-ATP pyrophospohydrolase
4 = Phosphoribosyl-AMP cyclohydrolase
5 = ProFAR-I (N’-[(5’phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase)
6 = Imidazole glycerol-phosphate synthase (IGPS)
7 = Ιmidazole glycerol-phosphate dehydratase
8 = Histidinol-phosphate aminotransferase
9 = Histidinol-phosphate phosphatase
10 = Histidinol dehydrogenase
1 - PRPP = Phosphoribosyl Pyrophosphate
2. PRATP = Phosphoribosyl ATP
3. PRAMP = Phosphoribosyl AMP
4. ProFAR = (N′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide) ribonucleotide
5. PRFAR = (N′-[(5-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide) ribonucleotide
6. IGP = Imidazole glycerol-phosphate
7. AICAR = 5′-phosphoribosyl-4-carboximide-5-aminoimidazole
8. IAP = Imidazole acetol-phosphate
9. α-KG = α-ketoglutarate
Histidine is a feedback inhibitor of ATP-phosphoribosyltransferase and thus helps to regulate its own synthesis. Histidine is the only amino acid to contain an imidazole ring. It is ionizable and has a pKa of about 6. As a result, histidine’s R-group can gain/lose a proton at pH values close to cellular conditions.
A cysteine analog commonly referred to as the 21st amino acid, selenocysteine (Figure 6.163) is an unusual amino acid occasionally found in proteins. Although it is rare, selenocysteine has been found in proteins in bacteria, archaea and eukaryotes.
In contrast to amino acids such as phosphoserine, hydroxyproline, or acetyl-lysine, which arise as a result of post-translational modifications, selenocysteine is actually built into growing peptide chains in ribosomes during the process of translation.
No codon specifies selenocysteine, so to incorporate it into a protein, a tRNA carrying it must bind to a codon that normally specifies STOP (UGA). This alternative reading of the UGA is dependent on formation of a special hairpin loop structure in the mRNA encoding selenoproteins.
Selenium is rather toxic, so cellular and dietary concentrations are typically exceedingly low. About 25 human proteins are known to contain the amino acid. These include five glutathione peroxidases, and three thioredoxin reductases. Iodothyronine deiodinase, a key enzyme that converts thyroxine to the active T3 form, also contains selenocysteine in its active site. All of these proteins contain a single selenocysteine.
A eukaryotic protein known as selenoprotein P, found in the blood plasma of animals, contains ten selenocysteine residues and is thought to function as an antioxidant and/or in heavy metal detoxification. Besides selenocysteine, at least two other biological forms of a seleno-amino acid are known. These include 1) selenomethionine (Figure 6.164), a naturally occurring amino acid in Brazil nuts, cereal grains, soybeans, and grassland legumes and 2) methylated forms of selenocysteine, such as Se-methylselenocysteine, are found in Astragalus, Allium, and Brassica species.
The specifics of the process of translation will be described elsewhere in the book, but to get selenocysteine into a protein, the tRNA carrying selenocysteine pairs with a stop codon (UGA) in the mRNA in the ribosome. Thus, instead of stopping translation, selenocysteine can incorporated into a growing protein and translation continues instead of stopping.
Four genes are involved in preparation of selenocysteine for incorporation into proteins. They are known as sel A, sel B, sel C, and sel D. Sel C codes for the special tRNA that carries selenocysteine. The amino acid initially put onto the selenocysteine tRNA is not selenocysteine, but rather serine. Action of sel A and sel D are necessary to convert the serine to a selenocysteine.
An intermediate in the process is selenophosphate, which is the selenium donor. It is derived from H2Se, the form in which selenium is found in the cell. The tRNA carrying selenocysteine has a slightly different structure than other tRNAs, so it requires assistance in translation. The sel B gene encodes for an EF-Tu-like protein that helps incorporate the selenocysteine into the protein during translation.
Recoding the UGA
Using UGA codons to incorporate selenocysteine into proteins could wreak havoc if done routinely, as UGA, in fact, almost always functions as a stop codon and is only rarely used to code for selenocysteine. Fortunately, there is a mechanism to ensure that the reading of a UGA codon as selenocysteine occurs only when the mRNA encodes a selenoprotein.
Unusual structures in mRNAs
The mRNAs for selenocysteine-containing proteins form unusual mRNA structures around the UGA codon that make the ribosome “miss” it as a stop codon and permit the tRNA with selenocysteine to be incorporated instead.
Like selenocysteine, pyrrolysine is a rare, unusual, genetically encoded amino acid found in some cells. Proteins containing it are enzymes involved in methane metabolism and so far have been found only methanogenic archaeans and one species of bacterium. The amino acid is found in the active site of the enzymes containing it. It is sometimes referred to as the 22nd amino acid.
Synthesis of the amino acid biologically begins with two lysines. One is converted to (3R)-3-Methyl-D-ornithine, which is attached to the second lysine. After elimination of an amine group, cyclization, and dehydration, L-pyrrolysine is produced. Pyrrolysine is attached to an unusual tRNA (pylT gene product) by action of the aminoacyl tRNA synthetase encoded by the pylS gene. This unusual tRNA can pair with the UAG stop codon during translation and allow for incorporation of pyrrolysine into the growing polypeptide chain during translation in a manner similar to incorporation of selenocysteine.
The urea cycle holds the distinction of being the first metabolic cycle discovered - in 1932, five years before the citric acid cycle. It is an important metabolic pathway for balancing nitrogen in the bodies of animals and it takes place primarily in the liver and kidney.
Organisms, like humans, that excrete urea are called ureotelic. Those that excrete uric acid (birds, for example) are called uricotelic and those that excrete ammonia (fish) are ammonotelic. Ammonia, of course, is generated by metabolism of amines and is toxic, so managing levels of it is critical for any organism. Excretion of ammonia by fish is one reason that an aquarium periodically requires cleaning and replacement of water.
Liver failure can lead to accumulation of nitrogenous waste and exacerbates the problem. As shown in Figure 1.166, the cycle contains five reactions, with each turn of the cycle producing a molecule of urea. Of the five reactions, three occur in the cytoplasm and two take place in the mitochondrion. (The reaction making carbamoyl phosphate, catalyzed by carbamoyl phosphate synthetase is not shown in the figure.)
Though the cycle doesn’t really have a starting point, a common place to begin discussion is with the molecule of ornithine. As discussed elsewhere in this book, ornithine intersects the metabolic pathways of arginine and proline.
Ornithine is found in the cytoplasm and is transported into the mitochondrion by the ornithine-citrulline antiport of the inner mitochonrial membrane. In the matrix of the mitochondrion, two reactions occur relevant to the cycle. The first is formation of carbamoyl phosphate from bicarbonate, ammonia, and ATP catalyzed by carbamoyl phosphate synthetase I.
Carbamoyl phosphate then combines with ornithine in a reaction catalyzed by ornithine transcarbamoylase to make citrulline.
The citrulline is transported out to the cytoplasm by the ornithine-citrulline antiport mentioned above. In the cytoplasm, citrulline combines with L-aspartate using energy of ATP to make citrullyl-AMP (an intermediate) followed by argininosuccinate. The reaction is catalyzed by argininosuccinate synthase.
Next, fumarate is split from argininosuccinate by argininosuccinate lyase to form arginine.
Water is used by arginase to cleave arginine into urea and ornithine, completing the cycle.
Urea is less toxic than ammonia and is released in the urine. Some organisms make uric acid for the same reason.
It is worth noting that aspartic acid, ammonia, and bicarbonate enter the cycle and fumarate and urea are produced by it. Points to take away include 1) ammonia is converted to urea using bicarbonate and the amine from aspartate; 2) aspartate is converted to fumarate which releases more energy than if aspartate were converted to oxaloacetate, since conversion of fumarate to malate to oxaloacetate in the citric acid cycle generates an NADH, but direct conversion of aspartate to oxaloacetate does not; and 3) glutamate and aspartate are acting as shuttles to funnel ammonia into the cycle. Glutamate, as will be seen below, is a scavenger of ammonia.
Urea cycle regulation
The urea cycle is controlled both allosterically and by substrate concentration. The cycle requires N-acetylglutamate (NAG) for allosteric activation of carbamoyl phosphate synthetase I. The enzyme that catalyzes synthesis of NAG, NAG synthetase, is activated by arginine and glutamate. Thus, an indicator of high amine levels, arginine, and an important shuttler of amine groups, glutamate, stimulates the enzyme that activates the cycle.
The reaction catalyzed by NAG synthetase is
At the substrate level, all of the other enzymes of the urea cycle are controlled by the concentrations of substrates they act upon. Only at high concentrations are the enzymes fully utilized.
Complete deficiency of any urea cycle enzyme is fatal at birth, but mutations resulting in reduced expression of enzymes can have mixed effects. Since the enzymes are usually not limiting for these reactions, increasing substrate can often overcome reduced enzyme amounts to a point by simply fully activating enzymes present in reduced quantities.
However, if the deficiencies are sufficient, ammonium can accumulate and this can be quite problematic, especially in the brain, where mental deficiencies or lethargy can result. Reduction of ammonium concentration relies on the glutamate dehydrogenase reaction (named for the reverse reaction).
Additional ammonia can be taken up by glutamate in the glutamine synthetase reaction.
The result of these reactions is that α-ketoglutarate and glutamate concentrations will be reduced and the concentration of glutamine will increase. For the brain, this is a yin/yang situation. Removal of ammonia is good, but reduction of α-ketoglutarate concentration means less energy can be generated by the citric acid cycle. Further, glutamate is, itself, an important neurotransmitter and a precursor of another neurotransmitter - γ-aminobutyric acid (GABA).
From an energy perspective, the urea cycle can be said to break even or generate a small amount of energy, if one includes the energy produced in releasing ammonia from glutamate (one NADH). There are two NADHs produced (including the one for converting fumarate to oxaloacetate), which give 4-6 ATPs, depending on how efficiently the cell performs electron transport and oxidative phosphorylation.
The cycle takes in 3 ATPs and produces 2 ADPs and one AMP. Since AMP is equivalent to 2 ATP, the cycle uses 4 ATP. Thus, the cycle either breaks even in the worst case or generates 2 ATPs in the best case.
Amino acid catabolism
Amino acids are divided according to the pathways involved in their degradation. There are three general categories. Ones that yield intermediates in the glycolysis pathway are called glucogenic and those that yield intermediates of acetyl-CoA or acetoacetate are called ketogenic. Those that involve both are called glucogenic and ketogenic. These are shown in Figures 6.167 and 6.168.
As seen in the two figures, amino acids largely produce breakdown products related to intermediates of the citric acid cycle or glycolysis, but this isn’t the complete picture. Some amino acids, like tryptophan, phenylalanine, and tyrosine yield hormones or neurotransmitters on further metabolism (as noted earlier). Others like cysteine and methionine must dispose of their sulfur and all of the amino acids must rid themselves of nitrogen, which can happen via the urea cycle, transamination, or both.
Breakdown of tyrosine (Figure 6.169) is a five step process that yields acetoacetate and fumarate. Enzymes involved include 1) tyrosine transaminase; 2) p-hydroxylphenylpyruvate dioxygenase; 3) homogentisate dioxygenase; 4) maleylacetoacetate cis-trans-isomerase; and 5) 4-fumaryl acetoacetate hydrolase.
Breakdown of leucine is a multi-step process ultimately yielding the ketone body acetoacetate and acetyl-CoA. Branched chain amino acids (BCAAs - valine, leucine, and isoleucine) rely on Branched Chain AminoTransferase (BCAT) followed by Branched Chain α-ketoacid dehydrogenase (BCKD) for catabolism.
Breakdown of isoleucine yields intermediates that are both ketogenic and glucogenic. These include acetyl-CoA and propionyl-CoA.
Breakdown of valine is a multi-step process ultimately yielding propionyl-CoA.