Metabolism of Nitrogenous Compound

Metabolism of Nitrogenous Compound

Mpenda F.N

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Introduction• This topic describes the amino acids that are

important in human nutrition. • It covers the digestion and absorbtion of proteins

in the gut.• The amino acid degradation pathways in the

tissues, and the urea cycle. • The amino acid synthesis pathways • There are separate topic dealing with

nucleotide and 'one carbon' metabolism and with porphyrin on the integration of metabolism.

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http://www.bmb.leeds.ac.uk/illingworth/metabol/nucleo.htm

http://www.bmb.leeds.ac.uk/illingworth/metabol/porphyr.htm

http://www.bmb.leeds.ac.uk/illingworth/metabol/shuttles.htm

Introduction…• Human proteins have very different lifetimes. • Total body protein is about 11kg, but about 25% of

this is collagen, which is metabolically inert. • A typical muscle protein might survive for three

weeks, but many liver enzymes turn over in a couple of days.

• Some regulatory enzymes have half-lives measured in hours or minutes.

• The majority of the amino acids released during protein degradation are promptly re-incorporated into fresh proteins. 3

Introduction…• Net protein synthesis accounts for less than one

third of the dietary amino acid intake, even in rapidly growing children consuming a minimal diet.

• Most of the ingested protein is ultimately oxidised to provide energy, and the surplus nitrogen is excreted, a little as ammonia but mostly as urea.

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Protein degradation• Soluble intracellular proteins are tagged for

destruction by attaching ubiquitin, a low molecular weight protein marker.

• They are then degraded in proteasomes to short peptides.

• A very few of these are displayed on the cell surface by the MHC [major histocompatibility] complex as part of the immune system,

• But most of them are further metabolised to free amino acids.

• Some proteins are degraded by an alternative system within the lysosomes.

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Protein degradation• Dietary proteins are initially denatured by the

stomach acid, in conjunction with limited proteolysis by pepsin.

• In young mammals gastric rennin partially hydrolyses and precipitates milk casein and increases gastric residence time.

• Gastric acid also kills most ingested bacteria, rendering the upper part of the gut almost sterile.

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• Protein digestion is largely completed in the small intestine at a slightly alkaline pH.

• The pancreatic proteases trypsin, chymotrypsin and elastase divide the proteins into short peptides.

• These are attacked from both ends by aminopeptidase and carboxypeptidase, and the fragments are finished off by dipeptidases secreted from the gut wall.

Protein degradation

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Amino acid catabolism• In animals, amino acids undergo oxidative

degradation in three different metabolic circumstances:During the normal synthesis and degradation of cellular

proteins.When a diet is rich in protein and the ingested amino

acids exceed the body’s needs for protein synthesis.During starvation or in uncontrolled diabetes mellitus,

when carbohydrates are either unavailable or not properly utilized, cellular proteins are used as fuel.

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Amino acid catabolism• The liver is the principal site of amino acid

metabolism.• Other tissues, such as the kidney, the small

intestine, muscles, and adipose tissue, take part. • The first step in the breakdown of amino acids is

the separation of the amino group from the carbon skeleton, usually by a transamination reaction.

• The carbon skeletons resulting from the deaminated amino acids are used to form either glucose or fats, or they are converted to a metabolic intermediate that can be oxidized by the citric acid cycle.

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Amino acid catabolism• Under all these metabolic conditions, amino acids

lose their amino groups to form α-keto acids, the “carbon skeletons” of amino acids.

• The α-keto acids undergo oxidation to CO2 and H2O • More importantly provide three- and four-carbon

units that can be converted by gluconeogenesis into glucose, the fuel for brain, skeletal muscle, and other tissues

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Amino acid catabolism• Therefore, the processes of amino acid degradation

converge on the central catabolic pathways.• The carbon skeletons of most amino acids finding their way to the citric acid cycle. • In some cases the reaction pathways of amino acid

breakdown closely parallel steps in the catabolism of fatty acids.

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Amino acid catabolism• The pathways for amino acid degradation include a key step in which the α-amino group is

separated from the carbon skeleton and shunted into the pathways of amino group metabolism

• We will deal first with amino group metabolism and nitrogen excretion.

• We will then deal with the fate of the carbon skeletons derived from the amino acids; along the way we see how the pathways are interconnected.

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Metabolic Fates of Amino Groups• Excess ammonia generated in other (extrahepatic)

tissues travels to the liver for conversion to the excretory form.

• Glutamate and glutamine play especially critical roles in nitrogen metabolism, acting as a kind of

general collection point for amino groups.• In the cytosol of hepatocytes, amino groups from

most amino acids are transferred to α-ketoglutarate to form glutamate, which enters mitochondria and gives up its amino group to form NH4.

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• Excess ammonia generated in most other tissues is converted to the amide nitrogen of glutamine, which passes to the liver, then into liver mitochondria.

• Glutamine or glutamate or both are present in higher concentrations than other amino acids in most tissues.

• In skeletal muscle, excess amino groups are generally

transferred to pyruvate to form alanine, another important molecule in the transport of amino groups to

the liver.

Metabolic Fates of Amino Groups

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Central role of glutamate• Glutamate also occupies a special position in amino acid

breakdown• Most of the nitrogen from dietary protein is ultimately

excreted from the body via the glutamate pool.• Glutamate is special because it is chemically related to 2-

oxoglutarate (= α-keto glutatarate) which is a key intermediate in the citric acid (Krebs) cycle.

• Glutamate can be reversibly converted into 2-oxoglutarate by transaminases or by glutamate dehydrogenase.

• In addition, glutamate can be reversibly converted into glutamine, an important nitrogen carrier, and the most common free amino acid in human blood plasma.

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Central role of glutamate

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• Because of the participation of 2-oxoglutarate in numerous transaminations, glutamate is a prominent intermediate in nitrogen elimination as well as in anabolic pathways.

• Glutamate, formed in the course of nitrogen elimination, is either oxidatively deaminated by liver glutamate dehydrogenase forming ammonia, or converted to glutamine by glutamine synthetase and transported to kidney tubule cells.

• There the glutamine is sequentially deamidated by glutaminase and deaminated by kidney glutamate dehydrogenase.


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• The ammonia produced in the latter two reactions is excreted as NH4

+ in the urine, where it helps maintain urine pH in the normal range of pH 4 to pH 8.

• The extensive production of ammonia by peripheral tissue or hepatic glutamate dehydrogenase is not feasible because of the highly toxic effects of circulating ammonia.

• Normal serum ammonium concentrations are in the range of 20–40μM, and an increase in circulating ammonia to about 400μM causes alkalosis and neurotoxicity.


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Urea biosynthesis occurs in four stages: Transamination oxidative deamination of glutamate ammonia transport reactions of the urea cycle

Biosynthesis of urea

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Transamination reactions• The first step in the catabolism of most L-amino

acids, once they have reached the liver, is removal of the α-amino groups, promoted by enzymes called aminotransferases or transaminases.

• In these transamination reactions, the -amino group is transferred to the -carbon atom of alpha-ketoglutarate, leaving behind the corresponding -keto acid analog of the amino acid

• The effect of transamination reactions is to collect the amino groups from many different amino acids in the form of L-glutamate

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• Transamination is an exchange of functional groups between any amino acid (except lysine, proline, and threonine) and an α-keto acid.

• The amino group is usually transferred to the keto carbon atom of pyruvate, oxaloacetate, or α-ketoglutarate, converting the α-keto acid to alanine, aspartate, or glutamate, respectively.

• Transamination reactions are catalyzed by specific transaminases (also called aminotransferases), which require pyridoxal phosphate as a coenzyme.

Transamination reactions

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•Inmanyaminotransferase reactions, α -ketoglutarate is the amino group acceptor.• All aminotransferases have pyridoxal phosphate (PLP) as cofactor. •The reaction is readily reversible.

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The role of Pyridoxal Phosphate in the Transfer of-Amino Groups to alpha-Ketoglutarate

• All aminotransferases have the same prosthetic group and the same reaction mechanism.

• The prosthetic group is pyridoxal phosphate (PLP), the coenzyme form of pyridoxine, or vitamin B6.

• Also we saw pyridoxal phosphate in as a coenzyme in the glycogen phosphorylase reaction.

• Its primary role in cells is in the metabolism of molecules with amino groups.

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• Pyridoxal phosphate functions as an intermediate carrier of amino groups at the active site of

aminotransferases.• It undergoes reversible transformations between its

aldehyde form, pyridoxal phosphate, which can accept an amino group, and its aminated form, pyridoxamine phosphate, which can donate its amino group to an α-keto acid.

The role of Pyridoxal Phosphate in the Transfer ofα-Amino Groups to α-Ketoglutarate

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L-GLUTAMATE DEHYDROGENASE OCCUPIES A CENTRAL POSITION IN NITROGEN METABOLISM

• Transfer of amino nitrogen to α-ketoglutarate forms L-glutamate.

• Release of this nitrogen as ammonia is then catalyzed by hepatic L-glutamate dehydrogenase• (GDH), which can use either NAD+ or NADP+• Conversion of α-amino nitrogen to ammonia by the concerted action of glutamate

aminotransferase and GDH is often termed “transdeamination”

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• The L-glutamate dehydrogenase reaction.

• NAD(P)+ means that either NAD+ or NADP+ can serve as co-substrate.

• The reaction is reversible but favors

• glutamate formation.


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• Glutamate dehydrogenase operates at an important intersection of carbon and nitrogen metabolism.

• An allosteric enzyme with six identical subunits, its activity is influenced by a complicated array of allosteric modulators.

• The best-studied of these are the positive modulator ADP and the negative modulator GTP

• Mutations that alter the allosteric binding site for GTP or otherwise cause permanent activation of glutamate dehydrogenase lead to a human genetic disorder called hyperinsulinism-hyperammonemia


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• Serum aminotransferases such as aspartate aminotransferase, AST (also called serum glutamate-oxaloacetate transaminase, SGOT) and alanine transaminase, ALT (also called serum glutamate-pyruvate transaminase (SGPT) have been used as clinical markers of tissue damage,

• with increasing serum levels indicating an increased extent of damage.

• As indicated earlier, ALT has an important function in the delivery of skeletal muscle carbon and nitrogen (in the form of alanine) to the liver in a series of reactoins referred to as the glucose-alanine cycle


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Glutamine Transports Ammonia in the Bloodstream

• Ammonia is quite toxic to animal ,and the levels present in blood are regulated.

• In many tissues, including the brain, some processes such as nucleotide degradation generate free ammonia.

• In most animals much of the free ammonia is converted to a nontoxic compound before export from the extrahepatic tissues into the blood and transport to the liver or kidneys.

• For this transport function, glutamate, critical to intracellular amino group metabolism, is supplanted by L-glutamine.• The free ammonia produced in tissues is combined with

glutamate to yield glutamine by the action of glutamine synthetase. 33


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• The glutamine is sequentially deamidated by glutaminase and deaminated by kidney glutamate dehydrogenase.

• The ammonia produced in the kidney reactions is excreted as NH4

+ in the urine.• This helps maintain urine pH in the normal range of pH 4 to

pH 8. • The extensive production of ammonia by peripheral tissue or

hepatic glutamate dehydrogenase is not feasible because of the highly toxic effects of circulating ammonia.

• Normal serum ammonium concentrations are in the range of 20–40μM, and an increase in circulating ammonia to about 400μM causes alkalosis and neurotoxicity.


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Glucose-alanine cycle• Alanine also plays a special role in transporting

amino groups to the liver in a nontoxic form, via a pathway called the glucose-alanine cycle

• In muscle and certain other tissues that degrade amino acids for fuel, amino groups are collected in the form of glutamate by transamination.

• Glutamate can transfer its -amino group to pyruvate, a readily available product of muscle glycolysis, by the action of alanine aminotransferase.

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• The alanine so formed passes into the blood and travels to the liver. • In the cytosol of hepatocytes alanine

aminotransferase transfers the amino group• from alanine to alpha-ketoglutarate, forming

pyruvate and glutamate

Glucose-alanine cycle

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Glucose-alanine cycle

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Urea cycle• About 80% of the excreted nitrogen is in the form

of urea is produced exclusively 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.

• In the urea cycle, amino groups of urea are donated by carbamoyl phosphate and aspartate, while the carbon atom of urea is contributed by bicarbonate.

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• 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.

Urea cycle

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Arginine

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• The net reaction for urea synthesis shows consumption of 4 "high energy" phosphoanhydride bonds, contributed by ATP.

• Two of these are used by for synthesis of carbamoyl phosphate from bicarbonate and ammonia.

• The ammonia is itself ultimately derived from various amino acids by the combined action of transaminase enzymes and glutamate dehydrogenase.

• Carbamoyl phosphate synthesis occurs in the mitochondrial matrix, and is catalyzed by carbamoyl phosphate synthetase I.

Urea cycle

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• The carbamoyl phosphate produced is then consumed in the synthesis of citrulline from ornithine.

• This reaction is catalyzed by ornithine carbamoyltransferase.

• The citrulline is shuttled out of the mitochondrion and into the cytosol, where the rest of the urea cycle takes place.

• Another amino acid-derived amino group is incorporated into the intermediate Aspartate is joined via its α-amino group to citrulline in the reaction catalyzed by argininosuccinate synthase to form argininosuccinate.

Urea cycle

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• The next reaction is the elimination of fumarate from argininosuccinate, yielding arginine.

• This step is catalyzed by argininosuccinase. • Finally, urea is produced by arginase, acting on

arginine and water as substrates. • Urea is secreted into the bloodstream, from which

it is ultimately eliminated by the kidneys for excretion.

• The ornithine produced in this last step is shuttled into the mitochondrial matrix, completing the cycle

Urea cycle

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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.

• Enzymes of the urea cycle are controlled at the gene level.

• long-term changes in the quantity of dietary protein, changes of 20-fold or greater in the concentration of cycle enzymes are observed.

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• 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 inactive in the absence of its obligate 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 synthase.

Regulation of the Urea Cycle

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Read on molecular basis of ammonia intoxication

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Krebs bicycle• Because the fumarate produced in the

argininosuccinase reaction is also an intermediate of the citric acid cycle.

• each cycle can operate independently and communication between them depends on the transport of key intermediates between the mitochondrion and cytosol.

• The fumarate generated in cytosolic arginine synthesis can therefore be converted to malate in the cytosol, and these intermediates can be further metabolized in the cytosol or transported into mitochondria for use in the citric acid cycle49

Urea Cycle Disorders (UCDs)• 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.

• Take your time read on UCDs

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• Aspartate formed in mitochondria by transamination between oxaloacetate and glutamate can be transported to the cytosol, where it serves as nitrogen donor in the urea cycle reaction catalyzed by argininosuccinate synthetase.

Krebs bicycle

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Catabolism of the Carbon Skeletonsof Amino Acids

• The 20 catabolic pathways converge to form only six major products, all of which enter the citric acid cycle.

• From here the carbon skeletons are diverted to gluconeogenesis or ketogenesis or are completely oxidized to CO2 and H2O.

• All or part of the carbon skeletons of seven amino acids are ultimately broken down to acetyl-CoA.

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• Five amino acids are converted to alpha-ketoglutarate, four to succinyl-CoA, two to fumarate, and two to oxaloacetate.

• Parts or all of six amino acids are converted to pyruvate, which can be converted to either acetyl-CoA or oxaloacetate.

• Note that some amino acids appear more than once, reflecting different fates for different parts of their carbon skeletons.

Catabolism of the Carbon Skeletonsof Amino Acids

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Some Amino Acids Are Converted to Glucose,Others to Ketone Bodies

• The seven amino acids that are degraded entirely or in part to acetoacetyl-CoA and/or acetyl-CoA—phenylalanine,tyrosine, isoleucine, leucine, tryptophan, threonine,lysine

• Can yield ketone bodies in the liver57

• Where acetoacetyl-CoA is converted to acetoacetate and then to acetone and -hydroxybutyrate .

• These are the ketogenic amino acids• Their ability to form ketone bodies is

particularly evident in uncontrolled diabetes mellitus, in which the liver produces large amounts of ketone bodies from both fatty acids and the ketogenic amino acids.


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• The amino acids that are degraded to pyruvate, - ketoglutarate, succinyl-CoA, fumarate, and/or oxaloacetate can be converted to glucose and glycogen.

• They are the glucogenic amino acids.• However, division between ketogenic and glucogenic

amino acids is not clear.• For examplel, five amino acids—tryptophan,

phenylalanine, tyrosine, threonine, and isoleucine—are both ketogenic and glucogenic

• Leucine is an exclusively ketogenic amino acid that is very common in proteins.


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Enzyme Cofactors Playing Important Roles inAmino Acid Catabolism

• We have already considered one important class: transamination reactions requiring pyridoxal

phosphate.• Another common type of reaction in amino acid

catabolism is one-carbon transfers, which usually involve one of three cofactors:biotin, tetrahydrofolate,or S-adenosylmethionine

Read on how these cofactors are synthesized60

Amino Acids Degraded to Pyruvate• The carbon skeletons of six amino acids are

converted in whole or in part to pyruvate.• The pyruvate can then be converted to either

acetyl-CoA (a ketone body precurs) or oxaloacetate (a precursor for gluconeogenesis).

• Thus amino acids catabolized to pyruvate are both ketogenic and glucogenic

• Tryptophan, cysteine, serine, glycine, and threonine

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• Portions of the carbon skeletons of seven amino acids—Tryptophan, lysine, phenylalanine, tyrosine, leucine,isoleucine, and threonine—yield acetyl-CoA and/or acetoacetyl-CoA, the latter being converted to acetyl-coA.

• Tryptophan breakdown is the most complex of all the pathways of amino acid catabolism in animal tissue

Amino Acids Degraded to Acetyl-CoA

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• Portions of tryptophan (four of its carbons) yield acetyl-CoA via acetoacetyl-CoA.

• Some of the intermediates in tryptophan catabolism are precursors for the synthesis of other biomolecules , including nicotinate, precursor of NAD and NADP in animals; serotonin.


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• The breakdown of phenylalanine is noteworthy .• Genetic defects in the enzymes of this pathway lead

to several inheritable human diseases.• Phenylalanine and its oxidation product tyrosine are

degraded into two fragments, both of which can enter the citric acid cycle:Four of the nine carbon atoms yield free

acetoacetate, which is converted to acetoacetyl-CoA.

Second four-carbon fragment is recovered as fumarate.


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• Phenylalanine, after its hydroxylation to tyrosine, is also the precursor of dopamine, a neurotransmitter, and of norepinephrine and epinephrine, hormones secreted by the adrenal medulla’.

• Melanin, the black pigment of skin and hair, is also derived from tyrosine.


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Phenylalanine catabolism is geneticallydefective in some people(Phenylketonuria: PKU)

• Phenylketonuria (PKU) is an autosomal recessive disorder resulting from defects in the metabolism of phenylalanine.

• PKU is a hyperphenylalaninemia with multifactorial causes. • There are several hyperphenylalaninemias that are not PKU

and are called non-PKU hyperphenylalaninemias (HPAs). • Hyperphenylalaninemia is defined as a plasma

phenylalanine concentration >120μM. • PKU is characterized by plasma phenylalanine >1000μM and

non-PKU hyperphenylalaninemias have plasma phenylalanine amounts that are <1000μM.

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• PKU is caused by mutation in the phenylalanine hydroxylase gene (gene symbol = PAH).

• The HPAs are disorders of phenylalanine hydroxylation. • The reaction catalyzed by PAH involves

tetrahydrobiopterin (BH4) as a co-factor, the HPAs can result from defects in any of the several genes required for synthesis and recycling of BH4.

• Removal of excess phenylalanine normally proceeds via the tyrosine biosynthesis reaction and then via tyrosine catabolism. The first reaction in this process is the PAH catalyzed hydroxylation of phenylalanine.70


• However, defects of other factors in phenylalanine hydroxylation system may have similar effect as phenylalanine hydroxylase defect.

• Also, hyperphenylalaninemia results in abnormalities in the metabolism of many compounds derived from the aromatic amino acids.

• If left untreated, these metabolic abnormalities cause postnatal brain damage and severe mental retardation.

• PKU is the most common inborn error in amino acid metabolism.

• The mutant gene frequency is such that 1 in 50 individuals is a carrier of the disease trait 71


• 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 2-oxoglutarate (α-ketoglutarate).

• This absence of 2-oxoglutarate 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


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• The carbon skeletons of proline, glutamate, glutamine, arginine, and histidine enter the citric

acid cycle as α-ketoglutarate. • Proline, glutamate, and glutamine have five-

carbon skeletons.• The cyclic structure of proline is opened by

oxidation to produce glutamate.

Amino Acids Converted to α-Ketoglutarate

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Amino Acids Converted to Succinyl-CoA• The carbon skeletons of methionine, isoleucine,

threonine, and valine are degraded by pathways that yield succinyl-CoA, an intermediate of the citric acid cycle.

• Methionine donates its methyl group to one of several possible acceptors through S-adenosylmethionine, and three of its four remaining carbon atoms are converted to the propionate of propionyl-CoA, a precursor of succinyl-CoA.

• Isoleucine undergoes transamination, followed by oxidative decarboxylation of the resulting α-keto acid.

• The remaining five-carbon skeleton is further oxidized to acetyl-CoA and propionyl-CoA.

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• Valine undergoes transamination and decarboxylation, then a series of oxidation reactions that convert the remaining four carbons to propionyl-CoA.

• Some parts of the valine and isoleucine degradative pathways closely parallel steps infatty acid degradation.

• In human tissues, threonine is also converted in two steps to propionyl-CoA. This is the primary pathway for threonine degradation in humans.

Amino Acids Converted to Succinyl-CoA

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Branched-Chain Amino Acids Are NotDegraded in the Liver

• Although much of the catabolism of amino acids takes place in the liver, the three amino acids with branched side chains (leucine, isoleucine, and valine) are oxidized as fuels primarily in muscle, adipose, kidney, and brain tissue.

• These extrahepatic tissues contain an aminotransferase, absent in liver, that acts on all three branched-chain amino acids to produce the corresponding-keto acids.

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Non-essential amino acid biosynthesisNon-essential amino acid• Alanine, Asparagine, Aspartate, Cysteine,

Glutamate, Glutamine, Glycine, Proline, Serine, Tyrosine.

Essential amino acid• Arginine, Histidine, Isoleucine, Leucine, Lysine,

Methionine, Phenylalanine, Threonine, Tyrptophan, Valine.

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Non-essential amino acid biosynthesis• The amino acids arginine, methionine and phenylalanine

are considered essential for reasons not directly related to lack of synthesis.

• Arginine is synthesized by mammalian cells but at a rate that is insufficient to meet the growth needs of the body and the majority that is synthesized is cleaved to form urea.

• Methionine is required in large amounts to produce cysteine if the latter amino acid is not adequately supplied in the diet.

• Similarly, phenylalanine is needed in large amounts to form tyrosine if the latter is not adequately supplied in the diet.

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Glutamate and Aspartate

•Glutamate is synthesized from its' widely distributed α-keto acid precursor by a simple one-step transamination reaction catalyzed by glutamate dehydrogenase (GDH). •As it has been discussed, the glutamate dehydrogenase reaction plays a central role in overall nitrogen homeostasis

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Like glutamate, aspartate is synthesized by a simple one-step transamination reaction catalyzed by aspartate aminotransferase, AST (formerly referred to as serum glutamate-oxalate transaminase, SGOT).

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Also, aspartate can be derived from asparagine through the action of asparaginase. The importance of aspartate as a precursor of ornithine for the urea cycle is described in the urea cycle.

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Alanine and the Glucose-Alanine Cycle

• Alanine is second only to glutamine in prominence as a circulating amino acid.

• 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.86

There are two main pathways to production of muscle alanine:

directly from protein degradation, and via the transamination of pyruvate by alanine transaminase, ALT (also referred to as serum glutamate-pyruvate transaminase, SGPT).

Alanine and the Glucose-Alanine Cycle

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Cysteine Biosynthesis: Role of Methionine• The sulfur for cysteine synthesis comes from the

essential amino acid methionine. • A condensation of ATP and methionine, catalyzed

by methionine adenosyltransferase (MAT), yields S-adenosylmethionine (SAM or AdoMet).

• 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.• MAT is also called S-adenosylmethionine

synthetase.

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• The synthesis of cysteine represents an extremely important and clinically relevant biochemical pathway.

• Several vitamins are required for this metabolic pathway to proceed emphasizing the nutritional impact.

• Folate, pyridoxal phosphate (PLP, B6), and B12 are all necessary for cysteine synthesis.

• The enzyme methionine synthase requires both folate and B12 for activity.

• Deficiency in either of these vitamins contributes to homocysteinemia and also to the development of macrocytic (megaloblastic) anemias.

• Cystathionine β-synthase is a PLP requiring enzyme demonstrating why B6 deficiency is also associated with the development of homocysteinemia

Cysteine Biosynthesis: Role of Methionine

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Homocysteinemia / Homocystinemia• Homocysteinemias (homocystinemias) represent a family of

inherited disorders resulting from defects in several of the genes involved in the conversion of methionine to cysteine.

• As the name implies, these disorders result in elevated levels of homocysteine and homocystine in the urine, where the elevated urine output of the metabolite is referred to as homocysteinurina.

• Homocystine is a disulfide-bonded homodimer of two homocysteines.

• This is similar to the formation of cystine from two cysteines. • In addition to homocysteinuria, patients excrete elevated

levels of methionine and metabolites of homocysteine

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B Vitamins and Homocysteine• Several studies have shown consistent and strong

relationships between low concentrations of blood folate, vitamin B12 and vitamin B6, and high concentrations of homocysteine which, in turn, have been linked with heart disease, stroke and other vascular outcomes.

• The breakdown of homocysteine to cysteine requires the vitamin B6 dependent enzyme, cystathionine beta synthase

• Re-methylation to methionine requires a vitamin B12 dependent enzyme, with folate as a cofactor.

• The most common cause of homocysteine accumulation, therefore, is deficiency or low availability of folate, vitamin B12 or vitamin B6. 93

• Active folate, known as 5-MTHF or 5-methyltetrahydrofolate, works in concert with vitamin B12 as a methyl-group donor in the conversion of homocysteine back to methionine.

• Normally, about 50% of homocysteine is remethylated; the remaining homocysteine is transsulfurated to cysteine, which requires vitamin B6 as a co-factor.

• This pathway yields cysteine, which is then used by the body to make glutathione, a powerful antioxidant that protects cellular components against oxidative damage. 94

B Vitamins and Homocysteine

• Vitamin B2 (riboflavin) and magnesium are also involved in homocysteine metabolism.

• Thus a person needs several different B-vitamins to help keep homocysteine levels low and allow for it to be properly transformed into helpful antioxidants like glutathione.

• Without B6, B12, B2, folate, and magnesium, dangerous levels of homocysteine may build up in the body.

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B Vitamins and Homocysteine

• Lowering high homocysteine is thought to be important because of its strong and consistent relationship with heart disease.

• High homocysteine has been linked with occurrence and mortality from heart disease - as well as with indicators of heart disease risk, including thickening of the carotid arteries and venous thrombosis.

• 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 disease96

Homocysteine and Heart Disease

How Elevated Homocysteine Leads to Vascular Damage

• If unhealthy levels of homocysteine accumulate in the blood, the delicate lining of an artery (endothelium) can be damaged.

• Homocysteine can both initiate and potentiate atherosclerosis.

• For example, homocysteine-induced injury to the arterial wall is one of the factors that can initiate the process of atherosclerosis, leading to endothelial dysfunction and eventually to heart attacks and strokes

• Several studies have shown that homocysteine can inflict damage to the arterial wall via multiple destructive molecular mechanisms. 97

Tyrosine Biosynthesis• Tyrosine is produced in cells by hydroxylating the

essential amino acid phenylalanine. • This relationship is much like that between cysteine

and methionine. • Half of the daily requirement for phenylalanine is

for the production of tyrosine; • If the diet is rich in tyrosine itself, the requirements

for phenylalanine can be reduced by about 50%.

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• Phenylalanine hydroxylase (PAH) is a mixed-function oxygenase: one atom of oxygen is incorporated into water and the other into the hydroxyl of tyrosine.

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

Tyrosine Biosynthesis

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• Missing or deficient phenylalanine hydroxylase results in hyperphenylalaninemia.

• Hyperphenylalaninemia is defined as a plasma phenylalanine concentration greater than 2mg/dL (120μM).

• The most widely recognized hyperphenylalaninemia (and most severe) is the genetic disease known as phenlyketonuria (PKU).

• Patients suffering from PKU have plasma phenylalanine levels >1000μM, whereas the non-PKU hyperphenylalaninemias exhibit levels of plasma phenylalanine <1000μM.

• Untreated PKU leads to severe mental retardation.

Tyrosine Biosynthesis

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Ornithine and Proline Biosynthesis• Glutamate serves as the precursor for the synthesis of

both ornithine and proline which are derived from the Δ1-pyrroline-5-carboxylate intermediate in the pathway.

• Formation of Δ1-pyrroline-5-carboxylate occurs via the action of the bi-functional enzyme, aldehyde dehydrogenase

• The transamination of the tautomeric form of Δ1-pyrroline-5-carboxylate (glutamate γ-semialdehyde) results in the generation of ornithine.

• The reduction of Δ1-pyrroline-5-carboxylate to proline occurs via the action of pyrroline-5-carboxylate reductase 1.

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Ornithine and Proline Biosynthesis

Serine biosynthesis

Serine can be derived from the glycolytic intermediate, 3-phosphoglycerate, in a three-step reaciton pathway. The first reaction is catalyzed by phosphoglycerate dehydrogenase (PHGDH). The second reaction is a simple transamination catalyzed by phosphoserine aminotransferase 1 (PSAT1) which utilizes glutamate as the amino donor and releases 2-oxoglutarate (α-ketoglutarate). The last step in the reaction pathway is catalyzed by phosphoserine phosphatase (PSPH).

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Glycine Biosynthesis• The main pathway to glycine is a one-step

reversible reaction catalyzed by serine hydroxymethyltransferase (SHMT).

• This enzyme is a member of the family of one-carbon transferases and is also known as glycine hydroxymethyltransferase.

• This reaction involves the transfer of the hydroxymethyl group from serine to the cofactor tetrahydrofolate (THF), producing glycine and N5,N10-methylene-THF

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Glycine Biosynthesis

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• Glycine is involved in many anabolic reactions other than protein synthesis including the synthesis of purine nucleotides, heme, glutathione, creatine and serine.

• Also, glycine functions in the central nervous system as an inhibitory neurotransmitter where it participates in regulating signals that process motor and sensory information that permit movement, vision and audition.

• Glycine is co-released with GABA which is the primary inhibitory neurotransmitter.

• Glycine action as a neurotransmitter is a function of the amino acid binding to a specific receptor, GlyR

Glycine BiosynthesisGlycine as a Neurotransmitter

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Aspartate/Asparagine and Glutamate/Glutamine Biosynthesis

Glutamate is synthesized by the reductive amination of 2-oxoglutarate (α-ketoglutarate) catalyzed by glutamate dehydrogenase it is thus a nitrogen-incorporating 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

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Aspartate is formed in a transamination 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

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•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 incorporating reaction.• Asparagine, however, is formed by an amidotransferase reaction.

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Specialized products of amino acids

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Specialized products of amino acids• Important products derived from amino acids include

heme, purines, pyrimidines, hormones, neurotransmitters, and biologically active peptides.

• Small peptides or peptide-like molecules not synthesized on ribosomes fulfill specific functions in cells.

• Histamine plays a central role in many allergic reactions. • Neurotransmitters derived from amino acids include γ-

aminobutyrate, 5-hydroxytryptamine (serotonin), dopamine, norepinephrine, and epinephrine.

• Many drugs used to treat neurologic and psychiatric conditions affect the metabolism,of these neurotransmitters.

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Tyrosine-Derived Neurotransmitters / Hormones

• Much of the tyrosine that does not get incorporated into proteins is catabolized for energy production.

• Another significant fate of tyrosine is conversion to the catecholamines.

• The catecholamines are dopamine, norepinephrine, and epinephrine.

• All three catecholamines exert effects in numerous locations in the body as either a neurotransmitter or as a hormone.

• Within the brain the catecholamines exert their effects as neurotransmitters, in the periphery they do so as hormones. 114

• Tyrosine is transported into catecholamine-secreting neurons and adrenal medullary cells where catecholamine synthesis takes place.

• The first step in the process requires tyrosine hydroxylase which, like phenylalanine hydroxylase (of tyrosine synthesis), requires tetrahydrobiopterin (H4B, or written as BH4) as cofactor.

• The tyrosine hydroxylase reaction represents the rate-limiting reaction of catecholamine biosynthesis.

• The dependence of tyrosine hydroxylase on H4B necessitates the coupling to the action of dihydropteridine reductase (DHPR) as is the situation for phenylalanine hydroxylase and tryptophan hydroxylase. 115


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• The product of the tyrosine hydroxylase reaction is 3,4-dihydrophenylalanine (L-DOPA; more commonly just DOPA).

• The enzyme DOPA decarboxylase then converts DOPA to dopamine.

• The enzyme dopamine β-hydroxylase then converts dopamine to norepinephrine.

• Dopamine β-hydroxylase is a major vitamin C and copper (Cu2+)-dependent enzyme.

• The last step of catecholamine biosynthesis is the conversion of norepinephrine to epinephrine which involves a methylation reaction. 116


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• The enzyme phenylethanolamine N-methyltransferase catalyzes this methylation reaction utilizing SAM as a methyl donor.

• In addition to epinephrine synthesis, the last reaction generates S-adenosylhomocysteine.

• Within the substantia nigra locus of the brain, and some other regions of the brain, synthesis proceeds only to dopamine.

• Within the locus coeruleus region of the brain the end product of the pathway is norepinephrine.

• Within adrenal medulla chromaffin cells, tyrosine is converted to norepinephrine and epinephrine117


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• The actions of norepinephrine and epinephrine are exerted via receptor-mediated signal transduction events.

• The receptors to which epinephrine and norepinephrine bind are referred to as adrenergic receptors.

• The adrenergic receptors are members of the G-protein coupled receptor (GPCR) family.

• There are two distinct classes of adrenergic receptor identified as the α (alpha) and β (beta) receptors.

• There are two distinct subclasses of α adrenergic receptor identified as the α1 and α2 sub-classes.

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• The α1 class contains the α1A, α1B, and α1D receptors.

• The α1 receptor class are coupled to Gq-type G-proteins that activate PLCβ resulting in increases in IP3 and DAG release from membrane PIP2.

• The α2 class contains the α2A, α2B, and α2C receptors.

• The α2 class of adrenergic receptors are coupled to G i-type G-proteins that inhibit the activation of adenylate cyclase and therefore, receptor activation results in reduced levels of cAMP and consequently reduced levels of active PKA.

• The β class of receptors is composed of three subtypes: β1, β2, and β3 each of which couple to Gs-type G-proteins resulting in activation of adenylate cyclase and increases in cAMP with concomitant activation of PKA. 120


• Dopamine binds to dopamineric receptors identified as D-type receptors and there are five subclasses identified as D1, D2, D3, D4, and D5.

• All five dopamine receptors belong the the G-protein coupled receptor (GPCR) family.

• The D1 and D5 dopamine receptors are coupled to the activation of Gs-type G-proteins and, therefore, receptor activation results in activation of adenylate cyclase.

• The D2, D3, and D4 dopamine receptors are coupled to Gi-type G-proteins and, therefore, receptor activation results in the inhibition of adenylate cyclase.

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Tryptophan-Derived Neurotransmitters• Tryptophan serves as the precursor for the

synthesis of serotonin (5-hydroxytryptamine, 5-HT) and melatonin (N-acetyl-5-methoxytryptamine)

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serotonin• Serotonin is synthesized through a two-step process

involving a tetrahydrobiopterin-dependent hydroxylation reaction (catalyzed by tryptophan-5-monooxygenase, also called tryptophan hydroxylase) and then a decarboxylation catalyzed by aromatic L-amino acid decarboxylase.

• Tryptophan hydroxylase represents the rate-limiting step in serotonin and melatonin synthesis.

• READ ON THE FUNCTIONS OF SEROTONIN

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Melatonin• 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. 124

• The effects of norepinephrine are exerted through interaction with β-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.

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Melatonin

Creatine Biosynthesis• Creatine synthesis begins in the kidneys using the amino

acids arginine and glycine. • The formation of guanidinoacetate from these two

amino acids is catalyzed by the enzyme glycine amidinotransferase, also called L-arginine:glycine amidinotransferase.

• Guanidinoacetate is transported to the blood and picked up by heptocytes where it is methylated forming creatine.

• The methyl donor for this reation is S-adenosylmethionine (SAM) and the reaction is catalyzed by the enzyme guanidinoacetate N-methyltransferase.

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• Creatine is released to the blood where is is picked up by the brain and skeletal muscle cells through the action of the transporter.

• Within these cells creatine is phosphorylated by creatine kinases (CK; also called creatine phosphokinase, CPK) that generate the high-energy storage compound, creatine phosphate.

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Creatine Biosynthesis

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• 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.

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Creatine Biosynthesis

Glutathione • Glutathione (GSH) is a tripeptide composed of glutamate,

cysteine and glycine. • Glutathione serves as a potent reductant eliminating

hydroxy radicals, peroxynitrites, and hydroperoxides.• It is conjugated to drugs to make them more water

soluble.• It is involved in amino acid transport across cell

membranes (the γ-glutamyl cycle)• It is a substrate for the peptidoleukotrienes.• it serves as a cofactor for some enzymatic reactions.• It serves as an aid in the rearrangement of protein disulfide

bonds.130

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• GSH is synthesized in the cytosol of all mammalian cells via the two-step reaction.

• The rate of GSH synthesis is dependent upon the availability of cysteine and the activity of the rate-limiting enzyme, γ-glutamylcysteine synthetase (also called glutamate-cysteine ligase, GCL).

• The second reaction of GSH synthesis involves the enzyme, glutathione synthetase, which condenses γ-glutamylcysteine with glycine.

• Both reactions of GSH synthesis require ATP.

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Glutathione

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Metabolism of Nitrogenous Compound