Investigation of detoxification processes and biosynthesis of urea. Hormonal adjusting and pathologies of proteins metabolism
Ammonia the highly toxic product of protein catabolism, is rapidly inactivated by a variety of reactions. Some product of these reactions are utilized for other purposes (thus salvaging a portion of the amino nitrogen), while others are excreted. The excreted form varies quite widely among vertebrate and invertebrate animals. The development of a pathway for nitrogen disposal in a species appears to depend chiefly on the availability of water.
Humans are totally dependent on other organisms for converting atmospheric nitrogen into forms available to the body. Nitrogen fixation is carried out by bacterial nitrogenases forming reduced nitrogen, NH4+ which can then be used by all organisms to form amino acids.
Overview of the flow of nitrogen in the biosphere. Nitrogen, nitrites and nitrates are acted upon by bacteria (nitrogen fixation) and plants and we assimilate these compounds as protein in our diets. Ammonia incorporation in animals occurs through the actions of glutamate dehydrogenase and glutamine synthase. Glutamate plays the central role in mammalian nitrogen flow, serving as both a nitrogen donor and nitrogen acceptor.
Reduced nitrogen enters the human body as dietary free amino acids, protein, and the ammonia produced by intestinal tract bacteria. A pair of principal enzymes, glutamate dehydrogenase and glutamine synthatase, are found in all organisms and effect the conversion of ammonia into the amino acids glutamate and glutamine, respectively. Amino and amide groups from these 2 substances are freely transferred to other carbon skeletons by transamination and transamidation reactions.
Aminotransferases exist for all amino acids except threonine and lysine. The most common compounds involved as a donor/acceptor pair in transamination reactions are glutamate and α-KG, which participate in reactions with many different aminotransferases. Serum aminotransferases such as aspartate aminotransferase, AST and alanine transaminase, ALT have been used as clinical markers of tissue damage, with increasing serum levels indicating an increased extent of damage. Alanine transaminase has an important function in the delivery of skeletal muscle carbon and nitrogen (in the form of alanine) to the liver. In skeletal muscle, pyruvate is transaminated to alanine, thus affording an additional route of nitrogen transport from muscle to liver. In the liver, alanine transaminase transfers the ammonia to α-KG and regenerates pyruvate. The pyruvate can then be diverted into gluconeogenesis. This process is referred to as the glucose-alanine cycle.
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. 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.
The reaction catalyzed by glutamate dehydrogenase is:
The glutamate dehydrogenase utilizes both nicotinamide nucleotide cofactors; NAD+ in the direction of nitrogen liberation and NADP+ for nitrogen incorporation. In the forward reaction as shown above glutamate dehydrogenase is important in converting free ammonia and α-KG to glutamate, forming one of the 20 amino acids required for protein synthesis. However, it should be recognized that the reverse reaction is a key anapleurotic process linking amino acid metabolism with TCA cycle activity. In the reverse reaction, glutamate dehydrogenase provides an oxidizable carbon source used for the production of energy as well as a reduced electron carrier, NADH. As expected for a branch point enzyme with an important link to energy metabolism, glutamate dehydrogenase is regulated by the cell energy charge. ATP and GTP are positive allosteric effectors of the formation of glutamate, whereas ADP and GDP are positive allosteric effectors of the reverse reaction. Thus, when the level of ATP is high, conversion of glutamate to α-KG and other TCA cycle intermediates is limited; when the cellular energy charge is low, glutamate is converted to ammonia and oxidizable TCA cycle intermediates. Glutamate is also a principal amino donor to other amino acids in subsequent transamination reactions. The multiple roles of glutamate in nitrogen balance make it a gateway between free ammonia and the amino groups of most amino acids.
The Glutamine Synthetase Reaction:
The glutamine synthetase reaction is also important in several respects. First it produces glutamine, one of the 20 major amino acids. Second, in animals, glutamine is the major amino acid found in the circulatory system. Its role there is to carry ammonia to and from various tissues but principally from peripheral tissues to the kidney, where the amide nitrogen is hydrolyzed by the enzyme glutaminase (reaction below); this process regenerates glutamate and free ammonium ion, which is excreted in the urine.
Note that, in this function, ammonia arising in peripheral tissue is carried in a non-ionizable form which has none of the neurotoxic or alkalosis-generating properties of free ammonia.
Liver contains both glutamine synthetase and glutaminase but the enzymes are localized in different cellular segments. This ensures that the liver is neither a net producer nor consumer of glutamine. The differences in cellular location of these two enzymes allows the liver to scavenge ammonia that has not been incorporated into urea. The enzymes of the urea cycle are located in the same cells as those that contain glutaminase. The result of the differential distribution of these two hepatic enzymes makes it possible to control ammonia incorporation into either urea or glutamine, the latter leads to excretion of ammonia by the kidney.
When acidosis occurs the body will divert more glutamine from the liver to the kidney. This allows for the conservation of bicarbonate ion since the incorporation of ammonia into urea requires bicarbonate (see below). When glutamine enters the kidney, glutaminase releases one mole of ammonia generating glutamate and then glutamate dehydrogenase releases another mole of ammonia generating α-KG. The ammonia will ionizes to ammonium ion (NH4+) which is excreted. The net effect is a reduction in the concentration of hydrogen ion, [H+], and thus an increase in the pH.
Earlier it was noted that kidney glutaminase was responsible for converting excess glutamine from the liver to urine ammonium. However, about 80% of the excreted nitrogen is in the form of urea which is also largely made in the liver, in a series of reactions that are distributed between the mitochondrial matrix and the cytosol. The series of reactions that form urea is known as the Urea Cycle or the Krebs-Henseleit Cycle.
1: carbamoyl phosphate synthetase-I (CPS-I)
2: ornithine transcarbamoylase (OTC)
3: argininosuccinate synthetase
The essential features of the urea cycle reactions and their metabolic regulation are as follows: arginine from the diet or from protein breakdown is cleaved by the cytosolic enzyme arginase, generating urea and ornithine. In subsequent reactions of the urea cycle a new urea residue is built on the ornithine, regenerating arginine and perpetuating the cycle.
Ornithine arising in the cytosol is transported to the mitochondrial matrix, where ornithine transcabamoylase catalyzes the condensation of ornithine with carbamoyl phosphate, producing citrulline. The energy for the reaction is provided by the high-energy anhydride of carbamoyl phosphate.
The product, citrulline, is then transported to the cytosol, where the remaining reactions of the cycle take place. The synthesis of citrulline requires a prior synthesis of carbamoyl phosphate (CP).
The activation step requires 2 equivalents of ATP and the mitochondrial matrix enzyme carbamoyl phosphate synthetase-I (CPS-I) (see reaction mechanism).There are two CP synthetases: a mitochondrial enzyme, CPS-I, which forms CP destined for inclusion in the urea cycle, and a cytosolic CP synthatase (CPS-II), which is involved in pyrimidine nucleotide biosynthesis. CPS-I is positively regulated by the allosteric effector N-acetyl-glutamate, while the cytosolic enzyme is acetylglutamate independent.
In a 2-step reaction, catalyzed by cytosolic argininosuccinate synthetase, citrulline is converted to argininosuccinate.
The reaction involves the addition of AMP (from ATP) to the amido carbonyl of citrulline, forming an activated intermediate on the enzyme surface (AMP-citrulline), and the subsequent addition of aspartate to form argininosuccinate.
Arginine and fumarate are produced from argininosuccinate by the cytosolic enzyme argininosuccinate lyase.
In the final step of the cycle arginase cleaves urea from aspartate, regenerating cytosolic ornithine, which can be transported to the mitochondrial matrix for another round of urea synthesis.
Beginning and ending with ornithine, the reactions of the cycle consumes 3 equivalents of ATP and a total of 4 high-energy nucleotide phosphates. Urea is the only new compound generated by the cycle; all other intermediates and reactants are recycled.
The energy consumed in the production of urea is more than recovered by the release of energy formed during the synthesis of the urea cycle intermediates. Ammonia released during the glutamate dehydrogenase reaction is coupled to the formation of NADH. In addition, when fumarate is converted back to aspartate, the malate dehydrogenase reaction used to convert malate to oxaloacetate generates a mole of NADH. These two moles of NADH, thus, are oxidized in the mitochondria yielding 6 moles of ATP.
Regulation of the Urea Cycle
The urea cycle operates only to eliminate excess nitrogen. On high-protein diets the carbon skeletons of the amino acids are oxidized for energy or stored as fat and glycogen, but the amino nitrogen must be excreted. To facilitate this process, enzymes of the urea cycle are controlled at the gene level. With long-term changes in the quantity of dietary protein, changes of 20-fold or greater in the concentration of cycle enzymes are observed. When dietary proteins increase significantly, enzyme concentrations rise. On return to a balanced diet, enzyme levels decline. Under conditions of starvation, enzyme levels rise as proteins are degraded and amino acid carbon skeletons are used to provide energy, thus increasing the quantity of nitrogen that must be excreted.
Short-term regulation of the cycle occurs principally at CPS-I, which is relatively inactive in the absence of its allosteric activator N-acetylglutamate. The steady-state concentration of N-acetylglutamate is set by the concentration of its components acetyl-CoA and glutamate and by arginine, which is a positive allosteric effector of N-acetylglutamate synthetase (glutamate transacylase).
A complete lack of any one of the enzymes of the urea cycle will result in death shortly after birth. However, deficiencies in each of the enzymes of the urea cycle, including N-acetylglutamate synthase, have been identified. These disorders are referred to as urea cycle disorders or UCDs. A common thread to most UCDs is hyperammonemia leading to ammonia intoxication with the consequences described below. Deficiencies in arginase do not lead to symptomatic hyperammonemia as severe or as commonly as in the other UCDs.
Clinical symptoms are most severe when the UCD is at the level of carbamoyl phosphate synthetase I. Symptoms of UCDs usually arise at birth and encompass, ataxia, convulsions, lethargy, poor feeding and eventually coma and death if not recognized and treated properly. In fact, the mortality rate is 100% for UCDs that are left undiagnosed. Several UCDs manifest with late-onset such as in adulthood. In these cases the symptoms are hyperactivity, hepatomegaly and an avoidance of high protein foods.
In general, the treatment of UCDs has as common elements the reduction of protein in the diet, removal of excess ammonia and replacement of intermediates missing from the urea cycle. Administration of levulose reduces ammonia through its action of acidifying the colon. Bacteria metabolize levulose to acidic byproducts which then promotes excretion of ammonia in the feces as ammonium ions, NH4+. Antibiotics can be administered to kill intestinal ammonia producing bacteria. Sodium benzoate and sodium phenylbutyrate can be administered to covalently bind glycine (forming hippurate) and glutamine (forming phenylacetylglutamine), respectively. These latter compounds, which contain the ammonia nitrogen, are excreted in the feces. Dietary supplementation with arginine or citrulline can increase the rate of urea production in certain UCDs.
Table of Urea Cycle Defects
Type I Hyperammonemia
Carbamoylphosphate synthetase I
with 24h - 72h after birth infant becomes lethargic, needs stimulation to feed, vomiting, increasing lethargy, hypothermia and hyperventilation; without measurement of serum ammonia levels and appropriate intervention infant will die: treament with arginine which activates N-acetylglutamate synthetase
N-acetylglutamate synthetase Deficiency
severe hyperammonemia, mild hyperammonemia associated with deep coma, acidosis, recurrent diarrhea, ataxia, hypoglycemia, hyperornithinemia: treatment includes administration of carbamoyl glutamate to activate CPS I
Type 2 Hyperammonemia
most commonly occurring UCD, only X-linked UCD, ammonia and amino acids elevated in serum, increased serum orotic acid due to mitochondrial carbamoylphosphate entering cytosol and being incorporated into pyrimidine nucleotides which leads to excess production and consequently excess catabolic products: treat with high carbohydrate, low protein diet, ammonia detoxification with sodium phenylacetate or sodium benzoate
episodic hyperammonemia, vomiting, lethargy, ataxia, siezures, eventual coma: treat with arginine administration to enhance citrulline excretion, also with sodium benzoate for ammonia detoxification
Argininosuccinate lyase (argininosuccinase)
episodic symptoms similar to classic citrullinemia, elevated plasma and cerebral spinal fluid argininosuccinate: treat with arginine and sodium benzoate
rare UCD, progressive spastic quadriplegia and mental retardation, ammonia and arginine high in cerebral spinal fluid and serum, arginine, lysine and ornithine high in urine: treatment includes diet of essential amino acids excluding arginine, low protein diet
Earlier it was noted that ammonia was neurotoxic. Marked brain damage is seen in cases of failure to make urea via the urea cycle or to eliminate urea through the kidneys. The result of either of these events is a buildup of circulating levels of ammonium ion. Aside from its effect on blood pH, ammonia readily traverses the brain blood barrier and in the brain is converted to glutamate via glutamate dehydrogenase, depleting the brain of α-ketoglutarate. As the α-ketoglutarate is depleted, oxaloacetate falls correspondingly, and ultimately TCA cycle activity comes to a halt. In the absence of aerobic oxidative phosphorylation and TCA cycle activity, irreparable cell damage and neural cell death ensue. In addition, the increased glutamate leads to glutamine formation. This depletes glutamate stores which are needed in neural tissue since glutamate is both a neurotransmitter and a precursor for the synthesis of α -aminobutyrate, GABA, another neurotransmitter. Therefore, reductions in brain glutamate affect energy production as well as neurotransmission.
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 α-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.
AMINO ACID BIOSYNTHESIS
Glutamate and Aspartate
Glutamate and aspartate are synthesized from their widely distributed α-keto acid precursors by simple transamination reactions. The former catalyzed by glutamate dehydrogenase and the latter by aspartate aminotransferase, AST. Aspartate is also derived from asparagine through the action of asparaginase. The importance of glutamate as a common intracellular amino donor for transamination reactions and of aspartate as a precursor of ornithine for the urea cycle.
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 glutamate-pyruvate aminotransferase.
The sulfur for cysteine synthesis comes from the essential amino acid methionine. A condensation of ATP and methionine catalyzed by methionine adenosyltransferase S-adenosylmethionine (SAM or AdoMet).
Biosynthesis of S-adenosylmethionine, SAM
SAM serves as a precurosor for numerous methyl transfer reactions the conversion of norepinephrine to epinenephrine. The result of methyl transfer is the conversion of SAM to S-adenosylhomocysteine. S-adenosylhomocysteine 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 methionine-sparing conditions and requires N5-methyl-tetrahydrofolate as methyl donor. This reaction was discussed in the context of vitamin B12-requiring enzymes. Transmethylation reactions employing SAM are extremely important, but in this case the role of S-adenosylmethionine 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 Pi 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 α-ketobutyrate. The sum of the latter two reactions is known as trans-sulfuration. Cysteine is used for protein synthesis and other body needs, while the α-ketobutyrate is decarboxylated and converted to propionyl-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.
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.
Biosynthesis of tyrosine from phenylalanine
Missing or deficient phenylalanine hydroxylase leads to the genetic disease known as phenlyketonuria (PKU), which if untreated leads to severe mental retardation. The mental retardation is caused by the accumulation of phenylalanine, which becomes a major donor of amino groups in aminotransferase activity and depletes neural tissue of α-ketoglutarate. This absence of α-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.
In other pathways, tetrahydrobiopterin is a cofactor. The effects of missing or defective dihydropteridine reductase cause even more severe neurological difficulties than those usually associated with PKU caused by deficient 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. The fate of glutamate semialdehyde depends on prevailing cellular conditions. Ornithine production occurs from the semialdehyde via a simple glutamate-dependent transamination, producing ornithine.
Ornithine synthesis from glutamate
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 pyrroline-5-carboxylate which is then reduced to proline by an NADPH-dependent reductase.
The main pathway to 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.
The main pathway to glycine is a 1-step reaction catalyzed by serine hydroxymethyltransferase.
This reaction involves the transfer of the hydroxymethyl group from serine to the cofactor tetrahydrofolate (THF), producing glycine and N5,N10-methylene-THF. Glycine produced from serine or from the diet can also be oxidized by 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.
Aspartate/Asparagine and Glutamate/Glutamine Biosynthesis
Glutamate is synthesized by the reductive amination of α-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 transamintion reaction catalyzed by aspartate transaminase, AST. This reaction uses the aspartate α-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 α-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.
PATHWAYS OF AMINO ACID DEGRADATION
There are 20 standard amino acids in proteins, with a variety of carbon skeletons. Correspondingly, there are 20 different catabolic pathways for amino acid degradation. In humans, these pathways taken together normally account for only 10 to 15% of the body's energy production. Therefore, the individual amino acid degradative pathways are not nearly as active as glycolysis and fatty acid oxidation. In addition, the activity of the catabolic pathways can vary greatly from one amino acid to the next, depending upon the balance between requirements for biosynthetic processes and the amounts of a given amino acid available. For this reason, we shall not examine them all in detail. The 20 catabolic pathways converge to form only five products, all of which enter the citric acid cycle. From here the carbons can be diverted to gluconeogenesis or ketogenesis, or they can be completely oxidized to CO2 and H2O.
All or part of the carbon skeletons of ten of the amino acids are ultimately broken down to yield acetyl-CoA. Five amino acids are converted into α-ketoglutarate, four into succinyl-CoA, two into fumarate, and two into oxaloacetate. The individual pathways for the 20 amino acids will be summarized by means of flow diagrams, each leading to a specific point of entry into the citric acid cycle. In these diagrams the amino acid carbon atoms that enter the citric acid cycle are shown in color. Note that some amino acids appear more than once, reflecting the fact that different parts of their carbon skeletons have different fates. Some of the enzymatic reactions in these pathways that are particularly noteworthy for their mechanisms or their medical significance will be singled out for special discussion.
Several Enzyme Cofactors Play Important Roles in Amino Acid Catabolism
A variety of interesting chemical rearrangements are found among the amino acid catabolic pathways. Before examining the pathways themselves, it is useful to note classes of reactions that recur and to introduce the enzymatic cofactors required. We have already considered one important class, the transamination reactions requiring pyridoxal phosphate. Another common type of reaction in amino acid catabolism is a one-carbon transfer. One-carbon transfers usually involve one of three cofactors: biotin, tetrahydrofolate, or S-adenosylmethionine.
These cofactors are used to transfer one-carbon groups in different oxidation states. The most oxidized state of carbon, CO2, is transferred by biotin. The remaining two cofactors are especially important in amino acid and nucleotide metabolism.
Tetrahydrofolate is generally involved in transfers of one-carbon groups in the intermediate oxidation states, and S-adenosylmethionine in transfers of methyl groups, the most reduced state of carbon.
Tetrahydrofolate (H4 folate) consists of a substituted pteridine, p-aminobenzoate, and glutamate linked together. This cofactor is synthesized in bacteria and its precursor, folate, is a vitamin for mammals. The one-carbon group, in any of three oxidation states, is bonded to N-5 or N-10 or to both. The most reduced form of the cofactor carries a methyl group, a more oxidized form carries a methylene group, and the most oxidized forms carry a methenyl, formyl, or formimino group. The different forms of tetrahydrofolate are interconvertible and serve as donors of one-carbon units in a variety of biosynthetic reactions. The major source of one-carbon units for tetrahydrofolate is the carbon removed in the conversion of serine to glycine, producing N5,N10-methylenetetrahydrofolate.
Although tetrahydrofolate can carry a methyl group at N-5, the methyl group's transfer potential is insufficient for most biosynthetic reactions. S-Adenosylmethionine is more commonly used for methyl group transfers. It is synthesized from ATP and methionine by the action of methionine adenosyl transferase. This reaction is unusual in that the nucleophilic sulfur atom of methionine attacks at the 5' carbon of the ribose moiety of ATP, releasing triphosphate, rather than attacking at one of the phosphorus atoms. The triphosphate is cleaved to Pi and PPi on the enzyme, and the PPi is later cleaved by inorganic pyrophosphatase, so that three bonds, two of which are high-energy bonds, are broken in this reaction. The only other reaction known in which triphosphate is displaced from ATP occurs in the synthesis of coenzyme B12.
S-Adenosylmethionine is a potent alkylating agent by virtue of its destabilizing sulfonium ion. The methyl group is subject to attack by nucleophiles and is about 1,000 times more reactive than the methyl group of N5-methyltetrahydrofolate.
Transfer of a methyl group from S-adenosylmethionine to an acceptor yields S-adenosylhomocysteine, which is subsequently broken down to homocysteine and adenosine. Methionine is regenerated by the transfer of a methyl group to homocysteine in a reaction catalyzed by methionine synthase. One form of this enzyme is common in bacteria and uses N5-methyltetrahydrofolate as a methyl donor. Another form that occurs in bacteria and mammals uses methylcobalamin derived from coenzyme B12. This reaction and the rearrangement of L-methylmalonyl-CoA to succinyl-CoA are the only coenzyme Bl2-dependent reactions known in mammals. Methionine is reconverted to S-adenosylmethionine to complete an activated methyl cycle.
Tetrahydrobiopterin is another cofactor introduced in these pathways, but it is not involved in one-carbon transfers. Tetrahydrobiopterin is structurally related to the flavin coenzymes, and it participates in biological oxidation reactions. It belongs to a widespread class of biological compounds called pterins, and we will consider its mode of action when we discuss phenylalanine degradation.
The carbon skeletons of ten amino acids yield acetyl-CoA, which enters the citric acid cycle directly. Five of the ten are degraded to acetyl-CoA via pyruvate. The other five are converted into acetyl-CoA and/or acetoacetyl-CoA, which is then cleaved to form acetyl-CoA.
The five amino acids entering via pyruvate are alanine, glycine, serine, cysteine, and tryptophan. In some organisms threonine is also degraded to form acetyl-CoA, in humans it is degraded to succinyl-CoA, as described later. Alanine yields pyruvate directly on transamination with a-ketoglutarate, and the side chain of tryptophan is cleaved to yield alanine and thus pyruvate. Cysteine is converted to pyruvate in two steps, one to remove the sulfur atom, the other a transamination. Serine is converted to pyruvate by serine dehydratase. Both the β-hydroxyl and the α-amino groups of serine are removed in this single PLP-dependent reaction. Glycine has two pathways. It can be converted into serine by enzymatic addition of a hydroxymethyl group. This reaction, catalyzed by serine hydroxymethyl transferase, requires the coenzymes tetrahydrofolate and pyridoxal phosphate. The second pathway for glycine, which predominates in animals, involves its oxidative cleavage into CO2, NH4+ , and a methylene group (-CH2-). This readily reversible reaction, catalyzed by glycine synthase, also requires tetrahydrofolate, which accepts the methylene group. In this oxidative cleavage pathway the two carbon atoms of glycine do not enter the citric acid cycle. One is lost as CO2, and the other becomes the methylene group of N5,N10- methylene- tetrahydrofolate, which is used as a one-carbon group donor in certain biosynthetic pathways.
Portions of the carbon skeleton of six amino acids-tryptophan, lysine, phenylalanine, tyrosine, leucine, and isoleucine-yield acetyl-CoA and/or acetoacetyl-CoA; the latter is then converted into acetyl-CoA. Some of the final steps in the degradative pathways for leucine, lysine, and tryptophan resemble steps in the oxidation of fatty acids. The breakdown of two of these six amino acids deserves special mention.
The catabolism of arginine begins within the context of the urea cycle. It is hydrolyzed to urea and ornithine by arginase.
The Arginine Metabolic Pathways: Nitric Oxide Synthase and Arginase
Two important metabolic pathways use the amino acid arginine as the precursor: the enzyme nitric oxide synthase, which converts arginine to nitric oxide, and citrulline and the enzyme arginase, which converts arginine to ornithine and urea. The latter is part of a pathway for detoxifying ammonia. Ornithine is also part of a proliferative pathway that is involved in cell division and tissue regeneration. Arginase II is the form of arginase that is thought to be involved in the synthesis of polyamines, which control cell proliferation and collagen production. It is most highly expressed in the prostate and kidney.1
There has been considerable recent publication of papers on nitric oxide synthase because of the importance of nitric oxide in functions such as (importantly) vasodilation (endothelial function). Scientists have found that an inadequate supply of arginine or too little of the cofactor tetrahydrobiopterin (which one paper reports may be mimicked by folic acid2) results in an “uncoupling” of nitric oxide synthase from the production of nitric oxide, producing superoxide anion instead. Not only is there a reduction in the production of nitric oxide when nitric oxide synthase is uncoupled, but oxidative stress is greatly increased.
Now, another major mechanism of decreased production of nitric oxide has been reported: an increase in the arginase pathway for the use of arginine. Recent studies have reported increases in arginase in conditions including reperfusion injury, asthma, psoriasis, arthritis, and human breast cancer. (Since arginase II is highly expressed in the prostate, it would be interesting to see whether there is increased expression in prostate cancer.) The increased arginase decreases arginine availability to be converted to nitric oxide, as well as increasing ornithine that can be converted into polyamines, procellular proliferation factors. In psoriasis, for example, there is hyperproliferation of keratinocytes. In the arthritis paper, it was reported that arginase II could be induced ex vivo (outside the body) by inflammatory factors such as PGE2 and LPS (lipopolysaccharide, from bacteria). Ornithine, produced by arginase, is necessary for the production of collagen, which occurs in rheumatoid arthritis.
Ornithine, in excess of urea cycle needs, is transaminated to form glutamate semialdehyde.
Nitric Oxide Synthases (NOS)
Is an important cellular-signaling molecule, a potent vasodilatator due to the smooth muscle relaxation. It also inhibits platelet adherence and aggregation, reduces adherence of leukocytes to the endothelium. Furthermore, NO has been shown to inhibit DNA synthesis and mitogenesis, and the proliferation of vascular smooth muscle cells. These antiproliferative effects are likely to be mediated by cyclic GMP.
Nitric Oxide Synthases from the biochemical point of view, are a family of complex enzymes catalyzing the oxidation of L-arginine to form NO and L-citrulline. The three human NOS isoforms identified to date are: eNOS (endothelial NOS), nNOS (neuronal NOS), and iNOS (inducible NOS). Their genes are found on human chromosomes 7, 12, and 17, respectively, and so they were named for the tissue in which they were first cloned and characterized. vasculoprotective effect of individual NOS isoforms in human organism is not sufficiently clarified yet. Endothelial NOS (eNOS) and neuronal NOS (nNOS) are constitutively expressed, mainly in endothelial cells and nitrergic nerves, respectively, synthesizing a small amount of NO under basal conditions and on stimulation by various agonists. By contrast, inducible NOS (iNOS) is expressed when stimulated by inflammatory stimuli, synthesizing a large amount of NO in a transient manner. The knowledge of nitric oxide synthases (NOSs) is of extreme scientific importance, not only for understanding new pathophysiological mechanisms but also as a target for therapeutic intervention.
The role of NO in regulating vascular tone and mediating platelet function is attributable to the ongoing activity of eNOS. It is pharmacologically identical with previously isolated EDRF (endothelium-derived releasing factor), exprimed by the intact endothelium. Inactivation of the eNOS pathway limits the contribution of NO to vessel homeostasis and results in increased vascular tone and platelet adhesion and aggregation. The activity of eNOS is regulated by the intracellular free calcium concentration and calcium- calmodulin complexes. Endothelial NOS is a constitutively expressed protein predominantly associated with the particulate subcellular fraction, suggesting that the native enzyme is a membrane-bound protein. A detailed analysis of the membrane association of eNOS showed that this enzyme is localized to the Golgi apparatus as well as to specific structures in the plasmalemmal membrane called caveolae. The association of eNOS with a region of the plasma membrane in which several key signal-transducing complexes are concentrated (such as G-proteins) is likely to have profound repercussions on enzyme activity as well as on its accessibility to intracellular mechanisms of the pathway release, including mechanisms independent of intracellular calcium release.
Neuronal Constitutive Nitric Oxide Synthase (nNOS) is present in central and peripheral neuronal cells and certain epithelial cells. Its activity is also regulated by Ca2+ and calmodulin. Its functions include long-term regulation of synaptic transmission in the central nervous system, central regulation of blood pressure, smooth muscle relaxation, and vasodilation via peripheral nitrergic nerves. It has also been implicated in neuronal death in cerebrovascular stroke. NO plays also an important role in the pathophysiology of some neurodegenerative diseases. The presence of NO and NOS should be proved indirectly through the histochemic positivity of nicotinamide dinucleotide phosphate diaphorase (NADPHd). It was proposed that nerve stimulation directly activated the release of NO from nitrergic nerves and, in fact, NO appears to be the dominant neurotransmitter responsible for the nerve-mediated, endothelium-independent vasodilation.
Glycine is classified as a glucogenic amino acid, since it can be converted to serine by serine hydroxymethyltransferase, and serine can be converted back to the glycolytic intermediate, 3-phosphoglycerate or to pyruvate by serine/threonine dehydratase.
Nevertheless, the main glycine catabolic pathway leads to the production of CO2, ammonia, and one equivalent of N5,N10-methyleneTHF by the mitochondrial glycine cleavage complex.
Hyperglycinemia refers to a condition where glycine is elevated in the blood.
- Propionic acidemia, also known as "ketotic glycinemia"
- Glycine encephalopathy, also known as "non-ketotic hyperglycinemia".
Glycine encephalopathy (also known as non-ketotic hyperglycinemia or NKH) is a rare autosomal recessive disorder of glycine metabolism. After phenylketonuria, glycine encephalopathy is the second most common disorder of amino acid metabolism. The disease is caused by defects in the glycine cleavage system, an enzyme responsible for glycine catabolism. There are several forms of the disease, with varying severity of symptoms and time of onset. The symptoms are exclusively neurological in nature, and clinically this disorder is characterized by abnormally high levels of the amino acid glycine in bodily fluids and tissues, especially the cerebral spinal fluid.
Glycine encephalopathy is sometimes referred to as "nonketotic hyperglycinemia" (NKH), as a reference to the biochemical findings seen in patients with the disorder, and to distinguish it from the disorders that cause "ketotic hyperglycinemia" (seen in propionic acidemia and several other inherited metabolic disorders). To avoid confusion, the term "glycine encephalopathy" is often used, as this term more accurately describes the clinical symptoms of the disorder. Glycine is metabolized in the body to end products of carbon dioxide and ammonia. The glycine cleavage system, which is responsible for glycine metabolism in the mitochondrion is made up of four protein subunits.
Propionic acidemia, also known as propionic aciduria, propionyl-CoA carboxylase deficiency and ketotic glycinemia, is an autosomal recessive metabolic disorder, classified as a branched-chain organic acidemia.
The disorder presents in the early neonatal period with progressive encephalopathy. Death can occur quickly, due to secondary hyperammonemia, infection, cardiomyopathy, or basal ganglial stroke.
Propionic Acidemia is a rare disorder that is inherited from both parents. Being autosomal recessive, neither parent shows symptoms, but both carry a defective gene responsible for this disease. It takes two faulty genes to cause PA, so there is a 1 in 4 chance for these parents to have a child with PA. Propionic acidemia is characterized almost immediately in newborns. Symptoms include poor feeding, vomiting, dehydration, acidosis, low muscle tone (hypotonia), seizures, and lethargy. The effects of propionic acidemia quickly become life-threatening.
Glutamine/Glutamate and Asparagine/Aspartate Catabolism
Glutaminase is an important kidney tubule enzyme involved in converting glutamine (from liver and from other tissue) to glutamate and NH3+, with the NH3+ being excreted in the urine. Glutaminase activity is present in many other tissues as well, although its activity is not nearly as prominent as in the kidney. The glutamate produced from glutamine is converted to -ketoglutarate, making glutamine a glucogenic amino acid. Asparaginase is also widely distributed within the body, where it converts asparagine into ammonia and aspartate. Aspartate transaminates to oxaloacetate, which follows the gluconeogenic pathway to glucose. Glutamate and aspartate are important in collecting and eliminating amino nitrogen via glutamine synthetase and the urea cycle, respectively. The catabolic path of the carbon skeletons involves simple 1-step aminotransferase reactions that directly produce net quantities of a TCA cycle intermediate. The glutamate dehydrogenase reaction operating in the direction of -ketoglutarate production provides a second avenue leading from glutamate to gluconeogenesis.
Alanine is also important in intertissue nitrogen transport as part of the glucose-alanine cycle. Alanine's catabolic pathway involves a simple aminotransferase reaction that directly produces pyruvate. Generally pyruvate produced by this pathway will result in the formation of oxaloacetate, although when the energy charge of a cell is low the pyruvate will be oxidized to CO2 and H2O via the PDH complex and the TCA cycle. This makes alanine a glucogenic amino acid.
The conversion of serine to glycine and then glycine oxidation to CO2 and NH3, with the production of two equivalents of N5,N10-methyleneTHF, was described above. Serine can be catabolized back to the glycolytic intermediate, 3-phosphoglycerate, by a pathway that is essentially a reversal of serine biosynthesis. However, the enzymes are different. Serine can also be converted to pyruvate through a deamination reaction catalyzed by serine/threonine dehydratase.
Glutamine is converted to glutamate by glutaminase or several other enzymes by the removal of the amide nitrogen. Proline is first converted to a Schiff base and then converted by hydrolysis to glutamate-5-semialdehyde. All of these changes occur on the same carbon. Arginine and histidine contain 5adjacent carbons and a sixth carbon attached through a nitrogen attom. The catabolism of these amino acids is thus slightly more complicated than glutamine or proline. Arginine is converted to ornithine and urea. Ornithine is furthere transaminated to produce glutamate-5-semialdehyde. Glutamate-5-semialdehyde is converted to glutamate. The enzymes involved in the steps of the histidine pathway are listed in the box in the lower right corner of the diagram. Tetrahydrofolate is the cofactor in the final step converting histidine to glutamate. Transamination or deamination of glutamate produces a-ketoglutarate which feeds into the citric acid cycle.
Glutamate semialdehyde can serve as the precursor for proline biosynthesis as described above or it can be converted to glutamate. Proline catabolism is a reversal of its synthesis process. The glutamate semialdehyde generated from ornithine and proline catabolism is oxidized to glutamate by an ATP-independent glutamate semialdehyde dehydrogenase. The glutamate can then be converted to ketoglutarate in a transamination reaction. Thus arginine, ornithine and proline, are glucogenic.
Hyperprolinemia is an excess of a particular protein building block (amino acid), called proline, in the blood. This condition generally occurs when proline is not broken down properly by the body. There are two inherited forms of hyperprolinemia, called type I and type II.
People with hyperprolinemia type I often do not show any symptoms, although they have proline levels in their blood between 3 and 10 times the normal level. Some individuals with hyperprolinemia type I exhibit seizures, intellectual disability, or other neurological or psychiatric problems.
Hyperprolinemia type II results in proline levels in the blood between 10 and 15 times higher than normal, and high levels of a related compound called pyrroline-5-carboxylate. This form of the disorder has signs and symptoms that vary in severity, and is more likely than type I to involve seizures or intellectual disability.
Hyperprolinemia can also occur with other conditions, such as malnutrition or liver disease. In particular, individuals with conditions that cause elevated levels of lactic acid in the blood (lactic acidemia) may have hyperprolinemia as well, because lactic acid inhibits the breakdown of proline.
Mutations in the ALDH4A1 and PRODH genes cause hyperprolinemia.
Inherited hyperprolinemia is caused by deficiencies in the enzymes that break down (degrade) proline. Hyperprolinemia type I is caused by a mutation in the PRODH gene, which provides instructions for producing the enzyme proline oxidase. This enzyme begins the process of degrading proline by starting the reaction that converts it to pyrroline-5-carboxylate.
Hyperprolinemia type II is caused by a mutation in the ALDH4A1 gene, which provides instructions for producing the enzyme pyrroline-5-carboxylate dehydrogenase. This enzyme helps to break down the pyrroline-5-carboxylate produced in the previous reaction, converting it to the amino acid glutamate. The conversion between proline and glutamate, and the reverse reaction controlled by different enzymes, are important in maintaining a supply of the amino acids needed for protein production, and for energy transfer within the cell.
A deficiency of either proline oxidase or pyrroline-5-carboxylate dehydrogenase results in a buildup of proline in the body. A deficiency of the latter enzyme leads to higher levels of proline and a buildup of the intermediate breakdown product pyrroline-5-carboxylate, causing the signs and symptoms of hyperprolinemia type II.
Histidine catabolism begins with release of the α-amino group catalyzed by histidase, introducing a double bond into the molecule. As a result, the deaminated product, urocanate, is not the usual α-keto acid associated with loss of α-amino nitrogens. The end product of histidine catabolism is glutamate, making histidine one of the glucogenic amino acids. Another key feature of histidine catabolism is that it serves as a source of ring nitrogen to combine with tetrahydrofolate (THF), producing the carbon THF intermediate known as N5-formiminoTHF. The latter reaction is one of two routes to N5-formiminoTHF. The principal genetic deficiency associated with histidine metabolism is absence or deficiency of the first enzyme of the pathway, histidase.
The resultant histidinemia is relatively benign. The disease, which is of relatively high incidence (1 in 10,000), is most easily detected by the absence of urocanate from skin and sweat, where it is normally found in relative abundance. Decarboxylation of histidine in the intestine by bacteria gives rise to histamine. Similarly, histamine arises in many tissues by the decarboxylation of histidine, which in excess causes constriction or dilation of various blood vessels. The general symptoms are those of asthma and various allergic reactions.
Histamine, biologically active substance found in a great variety of living organisms. It is distributed widely, albeit unevenly, throughout the animal kingdom and is present in many plants and bacteria and in insect venom. Histamine is chemically classified as an amine, an organic molecule based on the structure of ammonia (NH3). It is formed by the decarboxylation (the removal of a carboxyl group) of the amino acid histidine.
Histamine is synthesized in all tissues, but is particularly abundant in skin, lung and gastrointestinal tract. Mast cells, which are present in many tissues, are a prominent source of histamine, but histamine is also secreted by a number of other immune cells. Mast cells have surface receptors that bind immunoglobulin E, and when antigen crosslinks IgE on the mast cell surface, they respond by secreting histamine, along with a variety of other bioactive mediators.
Valine, Leucine and Isoleucine Catabolism
This group of essential amino acids are identified as the branched-chain amino acids, BCAAs. Because this arrangement of carbon atoms cannot be made by humans, these amino acids are an essential element in the diet. The catabolism of all three compounds initiates in muscle and yields NADH and FADH2 which can be utilized for ATP generation. The catabolism of all three of these amino acids uses the same enzymes in the first two steps. The first step in each case is a transamination using a single BCAA aminotransferase, with α-ketoglutarate as amine acceptor. As a result, three different α-keto acids are produced and are oxidized using a common branched-chain α-keto acid dehydrogenase, yielding the three different CoA derivatives. Subsequently the metabolic pathways diverge, producing many intermediates. The principal product from valine is propionylCoA, the glucogenic precursor of succinyl-CoA.
Isoleucine catabolism terminates with production of acetylCoA and propionylCoA; thus isoleucine is both glucogenic and ketogenic. Leucine gives rise to acetylCoA and acetoacetylCoA, and is thus classified as strictly ketogenic. There are a number of genetic diseases associated with faulty catabolism of the BCAAs. The most common defect is in the branched-chain α-keto acid dehydrogenase.
Since there is only one dehydrogenase enzyme for all three amino acids, all three α-keto acids accumulate and are excreted in the urine. The disease is known as Maple syrup urine disease because of the characteristic odor of the urine in afflicted individuals. Mental retardation in these cases is extensive. Unfortunately, since these are essential amino acids, they cannot be heavily restricted in the diet; ultimately, the life of afflicted individuals is short and development is abnormal The main neurological problems are due to poor formation of myelin in the CNS.
There are at least 3 pathways for threonine catabolism. One involves a pathway initiated by threonine dehydrogenase yielding a-amino-b-ketobutyrate. The α-amino-β-ketobutyrate is either converted to acetyl-CoA and glycine or spontaneously degrades to aminoacetone which is converted to pyruvate. The second pathway involves serine/threonine dehydratase yielding α-ketobutyrate which is further catabolized to propionyl-CoA and finally the TCA cycle intermediate, succinyl-CoA. The third pathway utilizes threonine aldolase. The products of this reaction are both ketogenic (acetyl-CoA) and glucogenic (pyruvate).
The principal fates of the essential amino acid methionine are incorporation into polypeptide chains, and use in the production of α-ketobutyrate and cysteine via SAM as described above. The transulfuration reactions that produce cysteine from homocysteine and serine also produce α-ketobutyrate, the latter being converted to succinyl-CoA. Regulation of the methionine metabolic pathway is based on the availability of methionine and cysteine.
If both amino acids are present in adequate quantities, SAM accumulates and is a positive effector on cystathionine synthase, encouraging the production of cysteine and α-ketobutyrate (both of which are glucogenic). However, if methionine is scarce, SAM will form only in small quantities, thus limiting cystathionine synthase activity. Under these conditions accumulated homocysteine is remethylated to methionine, using N5-methylTHF and other compounds as methyl donors.
In 1968, a Harvard researcher observed that children with a genetic defect that caused them to have sharply elevated homocysteine levels suffered severe atherosclerotic occlusion and vascular disorders similar to what is seen in middle-aged patients with arterial disease. This was the first indication that excess homocysteine might be an independent risk factor for heart disease.
There are several pathways for cysteine catabolism. The simplest, but least important pathway is catalyzed by a liver desulfurase and produces hydrogen sulfide, (H2S) and pyruvate. The more important catabolic pathway is via a cytochrome-P450-coupled enzyme, cysteine dioxygenase that oxidizes the cysteine sulfhydryl to sulfinate, producing the intermediate cysteinesulfinate. Cysteinesulfinate can serve as a biosynthetic intermediate undergoing decarboxylation and oxidation to produce taurine. Catabolism of cysteinesulfinate proceeds through transamination to b-sulfinylpyruvate which is in undergoes desulfuration yielding bisulfite, (HSO3-) and the glucogenic product, pyruvate. The enzyme sulfite oxidase uses O2 and H2O to convert HSO3- to sulfate, (SO4-) and H2O2. The resultant sulfate is used as a precursor for the formation of 3'-phosphoadenosine-5'-phosphosulfate,PAPS.
PAPS is used for the transfer of sulfate to biological molecules such as the sugars of the glycosphingolipids.Other than protein, the most important product of cysteine metabolism is the bile salt precursor taurine, which is used to form the bile acid conjugates taurocholate and taurochenodeoxycholate.The enzyme cystathionase can also transfer the sulfur from one cysteine to another generating thiocysteine and pyruvate. Transamination of cysteine yields -mercaptopyruvate which then reacts with sulfite, (SO32-), to produce thiosulfate, (S2O32-) and pyruvate. Both thiocysteine and thiosulfate can be used by the enzyme rhodanese to incorporate sulfur into cyanide, (CN-), thereby detoxifying the cyanide to thiocyanate.
Phenylalanine and Tyrosine Catabolism
Phenylalanine normally has only two fates: incorporation into polypeptide chains, and production of tyrosine via the tetrahydrobiopterin-requiring phenylalanine hydroxylase. Thus, phenylalanine catabolism always follows the pathway of tyrosine catabolism. The main pathway for tyrosine degradation involves conversion to fumarate and acetoacetate, allowing phenylalanine and tyrosine to be classified as both glucogenic and ketogenic. Tyrosine is equally important for protein biosynthesis as well as an intermediate in the biosynthesis of several physiologically important metabolites e.g. dopamine, norepinephrine and epinephrine.
As in phenylketonuria (deficiency of phenylalanine hydroxylase), deficiency of tyrosine transaminase leads to urinary excretion of tyrosine and the intermediates between phenylalanine and tyrosine.
Phenylketonuria (commonly known as PKU) is an inherited disorder that increases the levels of a substance called phenylalanine in the blood. Phenylalanine is a building block of proteins (an amino acid) that is obtained through the diet. It is found in all proteins and in some artificial sweeteners. If PKU is not treated, phenylalanine can build up to harmful levels in the body, causing intellectual disability and other serious health problems.
The signs and symptoms of PKU vary from mild to severe. The most severe form of this disorder is known as classic PKU. Infants with classic PKU appear normal until they are a few months old. Without treatment, these children develop permanent intellectual disability. Seizures, delayed development, behavioral problems, and psychiatric disorders are also common. Untreated individuals may have a musty or mouse-like odor as a side effect of excess phenylalanine in the body. Children with classic PKU tend to have lighter skin and hair than unaffected family members and are also likely to have skin disorders such as eczema.
Less severe forms of this condition, sometimes called variant PKU and non-PKU hyperphenylalaninemia, have a smaller risk of brain damage. People with very mild cases may not require treatment with a low-phenylalanine diet.
Babies born to mothers with PKU and uncontrolled phenylalanine levels (women who no longer follow a low-phenylalanine diet) have a significant risk of intellectual disability because they are exposed to very high levels of phenylalanine before birth. These infants may also have a low birth weight and grow more slowly than other children. Other characteristic medical problems include heart defects or other heart problems, an abnormally small head size (microcephaly), and behavioral problems. Women with PKU and uncontrolled phenylalanine levels also have an increased risk of pregnancy loss.
The adverse neurological symptoms are the same for the two diseases. Genetic diseases (such as various tyrosinemias and alkaptonuria) are also associated with other defective enzymes of the tyrosine catabolic pathway. The first genetic disease ever recognized, alcaptonuria, is caused by defective homogentisic acid oxidase. Homogentisic acid accumulation is relatively innocuous, causing urine to darken on exposure to air, but no life-threatening effects accompany the disease.
The other genetic deficiencies lead to more severe symptoms, most of which are associated with abnormal neural development, mental retardation, and shortened life span.
Albinism is a congenital disorder characterized by the complete or partial absence of pigment in the skin, hair and eyes due to absence or defect of tyrosinase, a copper-containing enzyme involved in the production of melanin. Albinism results from inheritance of recessive gene alleles and is known to affect all vertebrates, including humans. While an organism with complete absence of melanin is called an albino an organism with only a diminished amount of melanin is described as albinoid.
Albinism is associated with a number of vision defects, such as photophobia, nystagmus and astigmatism. Lack of skin pigmentation makes for more susceptibility to sunburn and skin cancers.
In rare cases such as Chédiak–Higashi syndrome, albinism may be associated with deficiencies in the transportation of melanin granules. This also affects essential granules present in immune cells leading to increased susceptibility to infection.
Lysine catabolism is unusual in the way that the α-amino group is transferred to α-ketoglutarate and into the general nitrogen pool. The reaction is a transamination in which the α-amino group is transferred to the α-keto carbon of α-ketoglutarate forming the metabolite, saccharopine. Unlike the majority of transamination reactions, this one does not employ pyridoxal phosphate as a cofactor. Saccharopine is immediately hydrolyzed by the enzyme α-aminoadipic semialdehyde synthase in such a way that the amino nitrogen remains with the α-carbon of α-ketoglutarate, producing glutamate and α-aminoadipic semialdehyde. Because this transamination reaction is not reversible, lysine is an essential amino acid. The ultimate end-product of lysine catabolism is acetoacetyl-CoA Genetic deficiencies in the enzyme α-aminoadipic semialdehyde synthase have been observed in individuals who excrete large quantities of urinary lysine and some saccharopine. The lysinemia and associated lysinuria are benign. Other serious disorders associated with lysine metabolism are due to failure of the transport system for lysine and the other dibasic amino acids across the intestinal wall. Lysine is essential for protein synthesis; a deficiencies of its transport into the body can cause seriously diminished levels of protein synthesis. Probably more significant however, is the fact that arginine is transported on the same dibasic amino acid carrier, and resulting arginine deficiencies limit the quantity of ornithine available for the urea cycle. The result is severe hyperammonemia after a meal rich in protein. The addition of citrulline to the diet prevents the hyperammonemia. Lysine is also important as a precursor for the synthesis of carnitine, required for the transport of fatty acids into the mitochondria for oxidation. Free lysine does not serve as the precursor for this reaction, rather the modified lysine found in certain proteins. Some proteins modify lysine to trimethyllysine using SAM as the methyl donor to transfer methyl groups to the α -amino of the lysine side chain. Hydrolysis of proteins containing trimethyllysine provide the substrate for the subsequent conversion to carnitine.
A number of important side reactions occur during the catabolism of tryptophan on the pathway to acetoacetate. The first enzyme of the catabolic pathway is an iron porphyrin oxygenase that opens the indole ring. The latter enzyme is highly inducible, its concentration rising almost 10-fold on a diet high in tryptophan. Kynurenine is the first key branch point intermediate in the pathway. Kynurenine undergoes deamniation in a standard transamination reaction yielding kynurenic acid. Kynurenic acid and metabolites have been shown to act as antiexcitotoxics and anticonvulsives. A second side branch reaction produces anthranilic acid plus alanine. Another equivalent of alanine is produced further along the main catabolic pathway, and it is the production of these alanine residues that allows tryptophan to be classified among the glucogenic and ketogenic amino acids. The second important branch point converts kynurenine into 2-amino-3-carboxymuconic semialdehyde, which has two fates. The main flow of carbon elements from this intermediate is to glutarate. An important side reaction in liver is a transamination and several rearrangements to produce limited amounts of nicotinic acid, which leads to production of a small amount of NAD+ and NADP+ Aside form its role as an amino acid in protein biosynthesis, tryptophan also serves as a precursor for the synthesis of serotonin and melatonin.
SPECIALIZED PRODUCTS OF AMINO ACIDS
The majority of tyrosine that does not get incorporated into proteins is catabolized for energy production. One other significant fate of tyrosine is conversion to the catecholamines. The catecholamine neurotransmitters are dopamine, norepinephrine, and epinephrine (see also Biochemistry of Nerve Transmission).Norepinephrine is the principal neurotransmitter of sympathetic postganglionic endings. Both norepinephrine and the methylated derivative, epinephrine are stored in synaptic knobs of neurons that secrete it, however, epinephrine is not a mediator at postganglionic sympathetic endings.Tyrosine is transported into catecholamine-secreting neurons and adrenal medullary cells where catechaolamine synthesis takes place. The first step in the process requires tyrosine hydroxylase, which like phenylalanine hydroxylase requires tetrahydrobiopterin as cofactor. The hydroxylation reaction generates DOPA (3,4-dihydrophenylalanine). DOPA decarboxylase converts DOPA to dopamine, dopamine b-hydroxylase converts dopamine to norepinephrine and phenylethanolamine N-methyltransferase converts norepinephrine to epinephrine. This latter reaction is one of several in the body that uses SAM as a methyl donor generating S-adenosylhomocysteine. Within the substantia nigra and some other regions of the brain, synthesis proceeds only to dopamine. Within the adrenal medulla dopamine is converted to norepinephrine and epinephrine.
Synthesis of the catecholamines from tyrosine
Once synthesized, dopamine, norepinephrine and epinephrine are packaged in granulated vesicles. Within these vesicles, norepinephrine and epinephrine are bound to ATP and a protein called chromogranin A. Metabolism of the catecholemines occurs through the actions of catecholamine-O-methyltransferase, (COMT) and monoamine oxidase, (MAO). Both of these enzymes are widley distributed throughout the body. However, COMT is not found in nerve endings as is MAO.
Tryptopan serves as the precursor for the synthesis of serotonin (5-hydroxytryptamine, 5-HT, see also Biochemistry of Nerve Transmission) and melatonin (N-acetyl-5-methoxytryptamine).
Serotonin is synthesized through 2-step process involving a tetrahydrobiopterin-dependent hydroxylation reaction (catalyzed by tryptophan-5-monooxygenase) and then a decarboxylation catalyzed by aromatic L-amino acid decarboxylase. The hydroxylase is normally not saturated and as a result, an increased uptake of tryptophan in the diet will lead to increased brain serotonin content. Serotonin is present at highest concentrations in platelets and in the gastrointestinal tract. Lesser amounts are found in the brain and the retina. Serotonin containing neurons have their cell bodies in the midline raphe nuclei of the brain stem and project to portions of the hypothalamus, the limbic system, the neocortex and the spinal cord. After release from serotonergic neurons, most of the released serotonin is recaptured by an active reuptake mechanism. The function of the antidepressant, Prozac is to inhibit this reuptake process, thereby, resulting in prolonged serotonin presence in the synaptic cleft. The function of serotonin is exerted upon its interaction with specific receptors. Several serotonin receptors have been cloned and are identified as 5HT1, 5HT2, 5HT3, 5HT4, 5HT5, 5HT6, and 5HT7. Within the 5HT1 group there are subtypes 5HT1A, 5HT1B, 5HT1D, 5HT1E, and 5HT1F. There are three 5HT2 subtypes, 5HT2A, 5HT2B, and 5HT2C as well as two 5HT5 subtypes, 5HT5a and 5HT5B. Most of these receptors are coupled to G-proteins that affect the activities of either adenylate cyclase or phospholipase C (PLC). The 5HT3 class of receptors are ion channels.Some serotonin receptors are presynaptic and others postsynaptic. The 5HT2A receptors mediate platelet aggregation and smooth muscle contraction. The 5HT2C receptors are suspected in control of food intake as mice lacking this gene become obese from increased food intake and are also subject to fatal seizures. The 5HT3 receptors are present in the gastrointestinal tract and are related to vomiting. Also present in the gastrointestinal tract are 5HT4 receptors where they function in secretion and peristalsis. The 5HT6 and 5HT7 receptors are distributed throughout the limbic system of the brain and the 5HT6 receptors have high affinity for antidepressant drugs. Melatonin is derived from serotonin within the pineal gland and the retina, where the necessary N-acetyltransferase enzyme is found. The pineal parenchymal cells secrete melatonin into the blood and cerebrospinal fluid. Synthesis and secretion of melatonin increases during the dark period of the day and is maintained at a low level during daylight hours. This diurnal variation in melatonin synthesis is brought about by norepinephrine secreted by the postganglionic sympathetic nerves that innervate the pineal gland. The effects of norepinephrine are exerted through interaction with b-adrenergic receptors. This leads to increased levels of cAMP, which in turn activate the N-acetyltransferase required for melatonin synthesis. Melatonin functions by inhibiting the synthesis and secretion of other neurotransmitters such as dopamine and GABA.
Creatine is synthesized in the liver by methylation of guanidoacetate using SAM as the methyl donor. Guanidoacetate itself is formed in the kidney from the amino acids arginine and glycine.
Synthesis of creatine and creatinine
Creatine is used as a storage form of high energy phosphate. The phosphate of ATP is transferred to creatine, generating creatine phosphate, through the action of creatine phosphokinase. The reaction is reversible such that when energy demand is high (e.g. during muscle exertion) creatine phosphate donates its phosphate to ADP to yield ATP.Both creatine and creatine phosphate are found in muscle, brain and blood. Creatinine is formed in muscle from creatine phosphate by a nonenzymatic dehydration and loss of phosphate. The amount of creatinine produced is related to muscle mass and remains remarkably constant from day to day. Creatinine is excreted by the kidneys and the level of excretion (creatinine clearance rate) is a measure of renal function.
Glutathione (abbreviated GSH) is a tripeptide composed of glutamate, cysteine and glycine that has numerous important functions within cells. It serves as a reductant, is conjugated to drugs to make them more water soluble, is involved in amino acid transport across cell membranes (the γ-glutamyl cycle), is a part of the peptidoleukotrienes, serves as a cofactor for some enzymatic reactions and as an aid in the rearrangement of protein disulfide bonds.
Synthesis of glutathione (GSH) Structure of GSSG
The role of GSH as a reductant is extremely important particularly in the highly oxidizing environment of the erythrocyte. The sulfhydryl of GSH can be used to reduce peroxides formed during oxygen transport. The resulting oxidized form of GSH consists of two molecules disulfide bonded together (abbreviated GSSG).
The enzyme glutathione reductase utilizes NADPH as a cofactor to reduce GSSG back to two moles of GSH. Hence, the pentose phosphate pathway is an extremely important pathway of erythrocytes for the continuing production of the NADPH needed by glutathione reductase. In fact as much as 10% of glucose consumption, by erythrocytes, may be mediated by the pentose phosphate pathway. Several mechanisms exist for the transport of amino acids across cell membranes. Many are symport or antiport mechanisms that couple amino acid transport to sodium transport. The γ-glutamyl cycle is an example of a group transfer mechanism of amino acid transport. Although this mechanism requires more energy input, it is rapid and has a high capacity. The cycle functions primarily in the kidney, particularly renal epithelial cells. The enzyme γ-glutamyl transpeptidase is located in the cell membrane and shuttles GSH to the cell surface to interact with an amino acid. Reaction with an amino acid liberates cysteinylglycine and generates a γ-glutamyl-amino acid which is transported into the cell and hydrolyzed to release the amino acid. Glutamate is released as 5-oxoproline and the cysteinylglycine is cleaved to its component amino acids. Regeneration of GSH requires an ATP-dependent conversion of 5-oxoproline to glutamate and then the 2 additional moles of ATP that are required during the normal generation of GSH.
One of the earliest signals that cells have entered their replication cycle is the appearance of elevated levels of mRNA for ornithine decarboxylase (ODC), and then increased levels of the enzyme, which is the first enzyme in the pathway to synthesis of the polyamines. Because of the latter, and because the polyamines are highly cationic and tend to bind nucleic acids with high affinity, it is believed that the polyamines are important participants in DNA synthesis, or in the regulation of that process.
The key features of the pathway are that it involves putrescine, an ornithine catabolite, and S-adenosylmethionine (SAM) as a donor of 2 propylamine residues. The first propylamine conjugation yields spermidine and addition of another to spermidine yields spermine.The function of ODC is to produce the 4-carbon saturated diamine, putrescine. At the same time, SAM decarboxylase cleaves the SAM carboxyl residue, producing decarboxylated SAM (S-adenosymethylthiopropylamine), which retains the methyl group usually involved in SAM methyltransferase activity. SAM decarboxylase activity is regulated by product inhibition and allosterically stimulated by putrescine. Spermidine synthase catalyzes the condensation reaction, producing spermidine and 5'-methylthioadenosine. A second propylamine residue is added to spermidine producing spermine.The signal for regulating ODC activity is unknown, but since the product of its activity, putrescine, regulates SAM decarboxylase activity, it appears that polyamine production is principally regulated by ODC concentration.The butylamino group of spermidine is used in a posttranslational modification reaction important to the process of translation. A specific lysine residue in the translational initiation factor eIF-4D is modified. Following the modification the residue is hydroxylated yielding a residue in the protein termed hypusine.
Nitric Oxide Synthesis and Function
Vasodilators, such as acetylcholine, do not exert their effects upon the vascular smooth muscle cell in the absence of the overlying endothelium. When acetylcholine binds its receptor on the surface of endothelial cells, a signal cascade, coupled to the activation phospholipase C (PLC), is initiated. The PLCg-mediated release of inositol trisphosphate, IP3 (from membrane associated phosphatidylinositol-4,5-bisphosphate, PIP2), leads to the release of intracellular stores of Ca2+. In turn, the elevation in Ca2+ leads to the liberation of endothelium-derived relaxing factor (EDRF) which then diffuses into the adjacent smooth muscle. Within smooth muscle cells, EDRF reacts with the heme moiety of a soluble guanylyl cyclase, resulting in activation of the latter and a consequent elevation of intracellular levels of cGMP. The net effect is the activation of cGMP-responsive enzymes which lead to smooth muscle cell relaxation. The coronary artery vasodilator, nitroglycerin, acts to increase intracellular release of EDRF and thus of cGMP. Quite unexpectedly, EDRF was found to be the free radical diatomic gas, nitric oxide, NO. NO is formed by the action of NO synthase, (NOS) on the amino acid arginine.
Nitric oxide is involved in a number of other important cellular processes in addition to its impact on vascular smooth muscle cells. Events initiated by NO that are important for blood coagulation include inhibition of platelet aggregation and adhesion and inhibition of neutrophil adhesion to platelets and to the vascular endothelium. NO is also generated by cells of the immune system and as such is involved in non-specific host defense mechanisms and macrophage-mediated killing. NO also inhibits the proliferation of tumor cells and microorganisms. Additional cellular responses to NO include induction of apoptosis (programmed cell death), DNA breakage and mutation. NOS is a very complex enzyme, employing five redox cofactors: NADPH, FAD, FMN, heme and tetrahydrobiopterin. NO can also be formed from nitrite, derived from vasodilators such as glycerin trinitrate during their metabolism. The half-life of NO is extremely short, lasting only 2-4 seconds. This is because it is a highly reactive free radical and interacts with oxygen and superoxide. NO is inhibited by hemoglobin and other heme proteins which bind it tightly.Chemical inhibitors of NOS are available and can markedly decrease production of NO. The effect is a dramatic increase in blood pressure due to vasoconstriction. Another important cardiovascular effect of NO is exerted through the production of cGMP, which acts to inhibit platelet aggregation.