*AMINO ACIDS 2GOALS/OBJECTIVESAt the end of this lecture students will be able to:Differentiate between protein and non-protein amino acids;Describe the functions of the different non-protein amino acids;Describe the processes of transamination and deamination;Describe the processes of the urea cycle and urea synthesis;

*Identify amino acid anabolic derivativesand their functions;Describe and differentiate amino acid-related diseases.

*NON-PROTEIN AMINO ACIDS.

The clinically significant non-protein amino acids include the following:

citrulline, ornithine, carnitine, creatine and taurine.

However, this list is not exhaustive and others do have some clinical significance, but usually in highly specialized areas or in minor pathways.

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*CITRULLINE

Free citrulline is mainly made from ornithine and carbamoyl phosphate in one of the central reactions in the urea cycle. Citrulline in proteins has been produced from arginine. Patients with rheumatoid arthritis often have detectable antibodies against proteins containing citrulline. Detection of antibodies reactive with citrulline-containing proteins or peptides is now becoming an important help in the diagnosis of rheumatoid arthritis. In recent studies, citrulline has been found to relax blood vessels. .

*ORNITHINE

Ornithine is a central part of the urea cycle, which allows for the disposal of excess nitrogen. First, ammonia is converted into carbamoyl phosphate onto which ornithine is added. Another nitrogen is added from aspartate, producing fumarate, and then arginine, which is hydrolysed back to ornithine, producing urea. In mammalian non-hepatic tissues, the main use of the urea cycle is in arginine biosynthesis, so as an intermediate in metabolic processes, ornithine is quite important.

*CARNITINE

Carnitine is synthesized primarily in the liver and kidneys from the amino acids lysine or methionine. Vitamin C (ascorbic acid) is essential to the synthesis of carnitine. Carnitine transports long-chain fatty acids into the mitochondrial matrix, so that they can be broken down through -oxidation to acetylCoA to obtain usable energy via the TCA cycle. Carnitine also has antioxidant properties, providing protection against lipid peroxidation of membranes.

*CREATINE

Creatine occurs in vertebrates and about half of the creatine originates from food, while the rest is synthesized from arginine, glycine, and methionine. It functions as creatine phosphate to provide short term energy. Creatinine is a break-down product of creatine phosphate and is usually produced at a fairly constant rate by the body. Chemically, creatinine is a cyclic derivative of creatine. Creatinine is filtered by the kidneys into the urine. There is no tubular reabsorption of creatinine. So, creatinine levels in blood and urine may be used to calculate the glomerular filtration rate (GFR).

*TAURINE

Taurine is a derivative of cysteine, and is conjugated with chenodeoxycholic acid and cholic acid to form the bile salts. Taurine crosses the blood-brain barrier and is an inhibitory neurotransmitter. It also acts as an antioxidant and protects against toxicity of various heavy metals. Taurine is thought to be a dietary essential nutrient in infants and has been added to many infant formulas.

*TRANSAMINATION AND DEGRADATION

AMINO ACIDS

GLUTAMATE

UREA

*Excess amino acids need to be removed from the body, but are not usually excreted in urine. Detection of amino acids in urine usually reflects either kidney or metabolic pathology. The prime mechanism for amino acid removal is for the amine group to be moved to another carbon backbone. This backbone is most often -ketoglutarate and the aminated product is glutamate.

This moving of the amine is termed transamination or aminotransfer and is usually the first step in their degradation. All the amino acids have specific transaminase enzymes which convert them to glutamate and their de-aminated -keto acid carbon backbone.

*

H | HOOC C NH2HOOC C = O | | X X

*All the transaminase enzymes require pyridoxal phosphate (vitamin B6) as coenzyme and during the reaction it accepts the amine from the amino acid, forming the -keto acid and becoming pyridoxamine, then the amine is donated to -ketoglutarate forming glutamate and pyridoxal phosphate again.

Since quantitatively alanine and aspartate are two of the most common amino acids, as well as being closely linked to energy metabolism, the transaminase enzymes that convert them to glutamate are the most common, especially in liver.

*

* COOH COOH l l CH2 CH2 l lCH3 CH2CH3 CH2l ll lCH-NH2+ C=OC=O + CH-NH2l ll lCOOH COOHCOOH COOH

* ASPARTATE + -KETOGLUTARATE OXALOACETATE + GLUTAMATE ASPARTATE TRANSAMINASE

COOH COOH l lCOOH CH2COOH CH2l ll lCH3 CH2CH3 CH2l ll lCH-NH2+ C=OC=O + CH-NH2l ll lCOOH COOHCOOH COOH

*This means that all amino acids can be converted to one common intermediate amino acid, which can then be metabolised and feed the amine into the urea cycle for urea synthesis. The -keto acid carbon backbones feed into the energy generation/fuel synthesis pathways at some point or other, depending on their structure.

The problem with this process is that -ketoglutarate levels in the cell are limited, so to keep the process going requires it to be regenerated. This is accomplished by linking the transaminase reactions to glutamate dehydrogenase, which converts glutamate back to -ketoglutarate and releases NH4+ for the urea cycle.

*Quantitatively, most of this process occurs in the liver, and the NH4+ is fed into the urea cycle for conversion to urea and ultimate excretion through the kidneys.

COOHCOOHllCH-NH2C=OllCH2+H20CH2+NH3/NH4+llCH2CH2llCOOHCOOHGLUTAMATE + WATER-KETOGLUTARATE + AMMONIA

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PUTTING IT ALL TOGETHER:

*Or another way of showing the same thing:

*A further effect of transamination is that the non-essential amino acids can to varying degrees be interconverted, as the carbon backbone can be synthesised and then an amine added via glutamate, and the appropriate transaminase, from another amino acid. This does not apply to the essential amino acids as humans cannot synthesise their carbon backbones.

Amino acids are classified as being either glucogenic or ketogenic. Glucogenic amino acids can be converted to an intermediate in glucose metabolism. Ketogenic amino acids can be converted to acetylCoA or acetoacetylCoA, but not glucose. Mixed amino acids can be converted to both a glucose intermediate as well as acetoacetylCoA or acetylCoA.

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Glucogenic amino acids are: aspartate, asparagine, glutamate, glutamine, histidine, proline, arginine, glycine, alanine, serine, cysteine, methionine and valine.

Ketogenic amino acids are: leucine and lysine.

Mixed amino acids are: phenylalanine, tyrosine, tryptophan, isoleucine and threonine.

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*UREA CYCLE

Urea is produced in the liver by a very efficient process that minimises the amount of carbon lost to transport the ammonia. As with most pathways the first step is the rate-limiting step, and produces carbamoyl phosphate from glutamate-derived ammonia and bicarbonate. The enzyme is carbamoyl phosphate synthetase, the reaction costs 2 ATP, and it occurs inside the mitochondria. Carbamoyl phosphate is then condensed with ornithine via ornithine transcarbamoylase and this produces citrulline, which then diffuses out of the mitochondria.In the cytosol, argininosuccinate synthetase combines citrulline with aspartate to form arginosuccinate. This also costs 1 ATP.

*Both the amine groups of urea are now in place, one from ammonium and one from aspartate. Arginosuccinase now cleaves the arginosuccinate to form arginine and fumarate. The fumarate recycles back to aspartate via oxaloacetate and can be reused in the cytosol. The arginine is then split via arginase to form urea and ornithine. The urea diffuses out of the liver cell and is transported in solution to the kidneys for excretion, the ornithine is retro-transported back into the mitochondria where the cycle can carry on again.

* NH4+ HCO3-MITOCHONDRIONCARBAMOYL PHOSPHATE

ORNITHINE

CITRULLINE

ASPARTATE

CYTOSOLARGININOSUCCINATE

OXALOACETATEARGININEFUMARATE

UREAPLASMA

* NH2

C=O

NH2

UREA

So the liver has taken a mixed pool of excess amino acids and converted them all to energy metabolism intermediates and a single excretory product, urea.

*The primary regulatory mechanism for the urea cycle is the concentration of the substrate for carbamoyl phosphate synthetase, the ammonia derived via glutamate deamination. The enzymes of the urea cycle are also induced or suppressed, depending on whether the diet is high or low in protein. Given the central role of the urea cycle in metabolism, defects in any of the enzymes are often fatal. Development in utero is usually normal, as the placenta and mother's circulation remove the excess ammonia. Post natal symptomology often develops rapidly and commonly causes early death.

*CORI, GLUCOSE-ALANINE CYCLEUnder aerobic conditions, ie. when the muscle is working, glycogenolysis and glycolysis provide substrates for the TCA cycle and ETC to make ATP for the muscle itself.When the muscle is resting, its energy requirements are very low, so the energy released from just glycolysis, ie. anaerobic, is adequate to fuel its requirements.However, this produces a lot of lactate which needs to be removed. It diffuses out of the cell into the circulation and goes to the liver. In the liver it is converted back to pyruvate, and this goes via gluconeogenesis to glucose. This is the Cori cycle.

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*Another cycle is going on at the same time. Under anaerobic conditions, protein turnover still continues, producing ammonia. This is converted to glutamate, which is then converted to alanine via transamination of some of the pyruvate produced from glycolysis. This alanine is then exported and sent to the liver, where it undergoes reverse transamination to produce pyruvate and glutamate again. The pyruvate then feeds into gluconeogenesis to produce glucose again, which recycles back to the muscle, while the glutamate feeds its ammonia into the urea cycle. Thus there is a continual recycling of 'waste products' from muscle to be recycled in the liver. This is the glucose-alanine cycle.

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*ALTALT

*HEME SYNTHESISThe synthesis of the heme group of hemoglobin, myoglobin and the cytochromes involves glycine directly, and several other amino acids indirectly.The process requires glycine to be condensed onto succinylCoA to form 5()-aminolevulinic acid (ALA). 2 of these form a dimer and then a pyrrole ring, porphobilinogen. 4 of these rings combine together to ultimately produce heme.SuccinylCoA can be produced from isoleucine, methionine and valine directly, or indirectly from glutamine, glutamate, histidine, proline and arginine via -ketoglutarate. SuccinylCoA is also a key intermediate in the TCA cycle, thus heme synthesis is closely linked to energy metabolism.

*SUCCINYLCoASUCCINATE

GLYCINEPORPHOBILINOGENPROTOPORPHYRINHEMOGLOBINFe2+

*AMINO ACID DERIVATIVES.

There are a range of derivatives of amino acids other than proteins and degradation products.

These fall into 2 categories:

Hormone derivatives, and neurotransmitter derivatives.

Hormone derivatives are made from tyrosine.

Neurotransmitter derivatives are, or are made from, glycine, aspartate, glutamate, choline, histidine, tyrosine and tryptophan.

*HORMONE DERIVATIVES.

Thyroid hormones (t3 and t4).Thyroid hormone synthesis starts with thyroglobulin. This is a large glycoprotein with 2 joined units. Iodine is incorporated into specific tyrosine residues at certain sites within the thyroglobulin structure. The iodinated thyroglobulin is then stored. When needed the thyroglobulin is degraded by lysosomal proteases. This produces either mono-iodotyrosine (MIT) or di-iodotyrosine (DIT). Lastly, the iodinated tyrosines are brought together to form the t3 or t4. 1 MIT + 1 DIT = t3, 1 DIT + 1 DIT = t4.

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*ADRENALINE.

Tyrosine is converted to dihydroxy-phenylalanine (DOPA) by tyrosine hydroxylase. This is then converted to dopamine by DOPA decarboxylase, and this dopamine converted to noradrenaline by dopamine--hydroxylase. Finally, adrenaline is produced via phenyl-ethanolamine-n-methyl transferase.

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*NEUROTRANSMITTERS.

Glycine, glutamate and aspartate function as neurotransmitters predominantly as themselves.

Glutamate is also converted to -amino butyric acid (GABA).

COOH

HOOC-CH2-CH2-CH-NH2 HOOC-CH2-CH2-CH2-NH2 GLUTAMATE DECARBOXYLASE

*HISTIDINE DECARBOXYLASEHistidine is converted to histamine.Choline is converted to acetylcholine.

*Tyrosine is converted to dopamine and noradrenaline.

*Tryptophan is converted to serotonin.

*AMINO ACID-RELATED DISEASESPhenylketonuria, alkaptonuria, albinism, maple syrup urine disease, ammonia toxicity, Parkinsonism, schizophrenia, cystinuria, MSG, urea cycle enzyme deficiency.

*PHENYLKETONURIA (PKU)This is an inborn error of metabolism with a deficiency of phenylalanine hydroxylase. The hydroxylation of phenylalanine is necessary in both normal degradation and the conversion of phenylalanine to tyrosine. The failure of this step leads to accumulation and excretion of other phenylalanine products, phenylpyruvate and phenyllactate. The deficiency causes severe mental retardation, minimal pigmentation, unusual gait, stance and sitting posture, and an increased frequency of epilepsy. The inability to synthesise tyrosine is the primary underlying cause of the cns problems, as dopamine and noradrenaline are deficient. Failure to synthesise melanin is also a direct result of tyrosine deficiency.

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*ALKAPTONURIA (BLACK URINE DISEASE)Homogentisic acid is a breakdown product of both phenylalanine and tyrosine and a deficiency of the enzyme homogentisic acid oxidase that oxidises this compound causes its accumulation and excretion in the urine. Homogentisic acid auto-oxidises and this gives the urine a very dark color, hence the common name. This is a rare condition, only about 1 in 1 million live births. It is not a particularly life-threatening condition, but can cause deposition of homogentisic acid into cartilage tissues, and this can lead to severe arthritis. There is no treatment other than to relieve the pain.

*MAPLE SYRUP URINE DISEASE (MSUD)The metabolism of leucine, isoleucine and valine initially removes the -amine group. This is followed by decarboxylation of the resulting -keto acid. This step is catalysed by branched-chain keto acid decarboxylase, an enzyme located inside the mitochondria. About 1 in 300 000 live births show this defect, and the effects are for them to pass the -keto acids in the urine, and these give a maple syrup smell. If untreated it can cause both physical and mental retardation. The only treatment is a low protein diet.

*CYSTINURIADifferent amino acids have different transport systems. A defect of the cysteine transporter system causes failure of reabsorption of cysteine from the glomerular filtrate. The unabsorbed cysteine oxidises to the dimer, cystine. Cystine is much less soluble than cysteine and tends to crystallise out, forming kidney stones. The common preventative treatment is to restrict intake of the cysteine precursor methionine, although the stones can be treated with ultrasound. Recently new drugs convert urinary cystine to a more soluble form.

*ALBINISMThis is reflection of the failure of the conversion of tyrosine to DOPA via tyrosinase and the subsequent failure to produce melanin. There does not appear to be a total lack of the enzyme, but rather a reduced capacity. The comparatively small amounts of DOPA required for neurotransmitter function can be produced, but the much greater amounts required for pigmentation are compromised. There is also evidence that the enzyme that converts DOPA to a melanin precursor quinone is deficient.

*HEREDITARY HYPERAMMONEMIAThis condition reflects a deficiency in ornithine transcarbamyolase, the key enzyme in condensing carbamoyl phosphate onto ornithine and allowing the urea cycle to clear excess ammonia as urea. Excess ammonia will cause increased levels of glutamate and glutamine, both of which will cause cns disturbance. Arginine supplementation can help to push the urea cycle faster as it is degraded to urea and ornithine.

*CHINESE RESTAURANT SYNDROME This condition is a reflection of excessive levels of glutamate in foods. The symptoms are a mixture of headache, nausea, sweating, weakness, facial flushes and tingling. Historically, it was associated with chinese food as soy sauce contains high levels of glutamate. It also underlies the concerns about monosodium glutamate food supplementation as similar effects have been reported. Severe asthmatics may suffer bronchospasm, but otherwise the effects reverse rapidly.

*PARKINSONISM (PARKINSONS DISEASE)This is a common condition in the elderly, although some young onset cases have been reported. It reflects progressive loss of dopaminergic neurons in the substantia nigra and locus ceruleus. Dopamine is an inhibitory neurotransmitter in the motor system, so uncontrollable tremor while resting is a characteristic symptom. Frequent cramping is also typical. It can be ameliorated initially by DOPA and a monoamine oxidase (MAO) inhibitor. The former is converted to dopamine, the latter prevents it breakdown. The condition is irreversible and progressive. The treatments also have side effects on other CNS areas where dopamine works.

*SCHIZOPHRENIAIt has been suggested that increased dopamine in the mesolimbic and mesocortical pathways may contribute to schizophrenia. Some of the most obvious evidence for this theory is from the effects amphetamine and cocaine. These drugs increase levels of dopamine in the brain and can cause effects which resemble those present in psychosis, particularly after large doses or prolonged use. This is often referred to as 'amphetamine psychosis' or 'cocaine psychosis' and may be virtually indistinguishable from schizophrenia.

*However, the acute effects of dopamine stimulants include euphoria, alertness and over-confidence; these symptoms are more reminiscent of mania than schizophrenia. Other studies showed that while some patients had over 90% of their dopamine D2 receptors blocked by antipsychotic drugs, they showed little reduction in their psychoses. Depending on the study, there was a spectrum of impact of dopaminergic antagonists on schizophrenia.

*THE END

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