Amino Acid Catabolism: Urea Cycle

Key steps in removal of the amino group

-3 key steps: transamination, oxidative deamination, and urea cycle -liver primary organ that performs the urea cycle

Urea cycle step 5

-Arginase hydrolyzes arginine to form ornithine and urea -excretion of urea removes two amino groups: one from carbamoyl phosphate and the other from aspartate Arginine --> Ornithine + Urea

Aspartate and alanine aminotransferases

-Aspartate aminotransferase (AST; also called glutamate oxaloacetate transaminase, GOT) catalyzes the transfer of the amino group of aspartate to a-ketoglutarate -Alanine aminotransferase (ALT; also called glutamate pyruvate transaminase, GPT) catalyzes the transfer of the amino group of alanine to a-ketoglutarate

Overview of catabolism

-Excess AAs cannot be stored or excreted -AAs in excess of the body's need are degraded to specific metabolic intermediates -major site of AA degradation is the liver

Treatment of urea cycle disorders

-Low protein diet: diet excluding arginine for arginase deficiency -Arginine supplementation: argininosuccinase deficiency -Sodium benzoate and phenyl butyrate administration: carbamoyl phosphate synthetase I deficiency; ornithine transcarbamoylase deficiency -N-Carbamoyl glutamate injection: N-Acetylglutamate synthase deficiency -Liver transplantation

N-acetylglutamate serves as an allosteric activator of CPS-I to enhance the urea cycle

-N-acetylglutamate is synthesized from glutamate and acetyl CoA by N-acetylglutamate synthase -activity of N-acetylglutamate is in turn stimulated by arginine, a metabolic intermediate of the urea cycle >>lots of arginine means lots of ammonium so activity of N-acetylglutamate synthase is enhanced

Degradation of amino acids

-Two parts: clearance of the amino group as urea and catabolism of the carbon skeletons -after AA is removed, carbon skeleton is converted into a major metabolite intermediate -AAs make a significant contribution to generation of energy in this way

How to differentiate b/w orotic acuduria caused by UMP synthase vs. OTC deficiency

-UMP synthase deficiency >>megaloblastic anemia, normal BUN & no hyperammonia -Ornithine transcarbamoylase deficiency >>hyperamoonemia, low BUN, & no megaloblastic anemia

Transamination overview

-a-amino group of an AA is transferred to a-ketoglutarate to form glutamate via a transamination reaction >>in this process, the carbon skeleton of the AA forms an a-ketoacid

Transamination is the first step in the removal of the amino group

-aminotransferase catalyzes the transfer of the a-amino group from an AA to an a-keto acid (generally a-ketoglutarate) >>this process requires pyridoxal phosphate (PLP) >>produces glutamate >>remaining carbon skeleton is also an a-keto acid -the remaining carbon skeleton of the AA is used for energy production while glutamate undergoes oxidative deamination to form NH4+ and regenerate a-ketoglutarate

Alanine cycle step 6

-ammonium enters the urea cycle and is converted to urea

Glutamine transports nitrogen from peripheral tissues to the liver (step 5)

-ammonium generated by glutaminase and glutamate dehydrogenase enters the urea cycle to form urea which is then excreted in the urine

OTC or CPS I defect shuts down the urea cycle

-arginine therapy, unfortunately, will not work in patients with ornithine transcarbamoylase or carbamoyl phosphate synthetase I deficiency >>these deficiencies will compromise the formation of citrulline -individuals with a deficiency in ornithine transcarbamoylase will have high levels of carbamoyl phosphate in mitochondria >>carbamoyl phosphate can diffuse out of mitochondria and gets utilized in the pyrimidine base biosynthesis in the cytoplasm to form orotic acid >>high levels of orotic acid will give rise to orotic aciduria -orotic acuduria can also arise if an individual has a deficiency in UMP synthase >>however, these individuals will not have hyperammonemia -individuals with transcarbamoylase deficiency will not have megaloblastic anemia

Urea cycle step 4

-argininosuccinase cleaves argininosuccinate into arginine and fumarate -fumarate formed from this reaction can be shunted into the TCA cycle Argininosuccinate --> Arginine + fumarate

Urea cycle step 3

-argininosuccinate synthetase catalyzes the formation of argininosuccinate from citrulline and aspartate -aspartate contributes an additional amino group into the urea cycle which is later removed as urea -citrulline transported to cytoplasm where it combines with aspartate to form argininosuccinate >>reaction requires hydrolysis of ATP into AMP and pyrophosphate Citrulline + Aspartate --> Argininosuccinate

What are major aminotransferases found in the heart and liver?

-aspartate and alanine aminotransferases

Why PLP is necessary

-basic mechanism of transamination involves the formation of a Schiff's base -amino group is transferred from the AA to the enzyme-bound pyridoxal phosphate (PLP) forming pyridoxamine phosphate (PMP) -PMP then donates the amino group to an a-keto acid (a-ketoglutarate) to form another AA

Urea cycle step 1

-carbamoyl phosphate synthetase I catalyzes first step -cycle begins with formation of carbamoyl phosphate from bicarbonate and ammonia in the mitochondria >>reaction consumes two molecules of ATP >>all three steps of this reaction catalyzed by CPS I -rate-limiting step in the urea cycle -CPS 1 is stimulated by N-acetylglutamate Bicarbonate --> carboxyphosphate (add ammonia)--> carbamic acid --> carbamoyl phosphate

Liver damage or urea cycle defects will result in hyperammonemia

-defects in the urea cycle lead to elevated NH4+, a toxic substance -high NH4+ levels in the brain will promote glutamine synthesis resulting in osmotic imbalance and brain damage -evident in some infants soon after birth; the afflicted infant becomes lethargic and vomits periodically; this is followed by coma and irreversible brain damage -signs and symptoms: lethargy, poor feeding, hypotonia, vomiting, hyperventilation, and seizures

Urea Cycle Defects

-disorder incidence is 1:30,000 -N-Acetylglutamate synthase deficiency -Carbamoyl phosphate synthetase I deficiency -Ornithine transcarbamoylase deficiency (orotic aciduria) -Argininosuccinate synthetase deficiency (citrullinemia) -Argininosuccinase deficiency -Arginase deficiency (arginemia) *disorder occurs when there is a deficiency in N-acetylglutamate synthase or one of the five enzymes in the urea cycle*

Alanine cycle step 5

-glutamate formed by the transamination reaction (conversion of alanine to pyruvate) then undergoes oxidative deamination to regenerate a-ketoglutarate and in the process ammonium is released

Glutamine transports nitrogen from peripheral tissues to the liver (step 1)

-glutamate formed from transamination reactions or nitrogen fixation is then acted on by glutamine synthetase

Alanine cycle step 1

-glutamate is formed by transamination reactions or by nitrogen fixation in the muscle

Oxidative deamination overview

-glutamate is oxidatively deaminated to yield an ammonium ion (NH4+) and to regenerate a-ketoglutarate

Glutamine transports nitrogen from peripheral tissues to the liver (step 4)

-glutamate is then oxidatively deaminated by glutamate dehydrogenase to produce a-ketoglutarate and ammonium

Glutamine transports nitrogen from peripheral tissues to the liver (step 3)

-glutamine is then released into the bloodstream and picked up by the liver -glutamine is first hydrolyzed by glutaminase to form glutamate and ammonium

Transport of extra hepatic nitrogen

-in extra hepatic tissues the AA degradation is processed similarly - first step is the removal of nitrogen from the amino acid -b/c skeletal muscle lacks the enzymes of the urea cycle, the nitrogen must be released in a neutral form (alanine and glutamine) that can be absorbed by the liver and converted to urea -urea is then released into the blood >>this is picked up by the kidneys and excreted in urine

Urea cycle basic

-in most terrestrial vertebrates, NH4+ is converted to urea via the urea cycle -urea is then excreted in the urine

Treatment of argininosuccinase deficiency with arginine

-ingestion of large amounts of arginine is recommended -arginine is converted to urea by arginase and in the process ornithine is generated -ornithine can react with carbamoyl phosphate to make citrulline -citrulline is then converted into argininosuccinate which can be excreted in the urine -arginine replenishes the supply of ornithine needed for the removal of carbamoyl phosphate -nitrogen is removed in this case, as argininosuccinate

Urea cycle overview

-involves 5 enzymes -ammonia and aspartate enter, and fumarate and urea leave the cycle -reactions take place in liver mitochondria and cytoplasm (2 mitochondrial and 3 cytoplasmic) -citrulline is made in mito. and transported out into the cytoplasm -ornithine is made in the cytoplasm and must be transported into the mito. -ornithine and citrulline are AAs but they are not used as protein building blocks

What is the use of aspartate and alanine aminotransferase measurement tests?

-measurement of the blood serum concentrations of these two enzymes (SGOT and SPGT tests) can provide information about severity of the damage caused by heart attack, liver damage, or infection -enzymes leak from injured cells -important in occupational medicine to determine whether workers exposed to toxic industrial solvents have suffered liver damage too

Alanine shuttle transports nitrogen from muscle to the liver

-nitrogen is transported from skeletal muscle to the liver in two principal transport forms (as alanine and glutamine) -this transport of nitrogen from skeletal muscle to the liver is referred to as the alanine cycle >>functions as a mech. to transport nitrogen from extra hepatic tissues to the liver

Urea cycle step 2

-ornithine transcarbamoylase converts ornithine and carbamoyl phosphate into citrulline -carbamoyl phosphate carries an amino group into the urea cycle Ornithine + carbamoyl phosphate --> citrulline

Ammonium can be produced by a number of processes in the body

-purine nucleotide catabolism -deamination of the side chain amino groups of glutamine and asparagine -direct deamination of histidine, serine, and threonine -degradation of urea and amino acids by gut bacteria -ability of glutamate dehydrogenase to fix ammonium reduces the unwanted buildup of ammonium which is toxic to cells at high levels

Alanine cycle step 4

-pyruvate (from alanine) is used for gluconeogenesis and the resulting glucose is released into the blood

Glutamate dehydrogenase carries out the oxidative deamination of glutamate to ammonium (NH4+)

-reaction catalyzed by glutamate dehydrogenase is reversible -can also be used to fix ammonia into organic molecules (reductive amination) -reaction regenerates a-ketoglutarate -ammonium ion can then be released for disposal as urea via the urea cycle -glutamate dehydrogenase found in mitochondria of almost all tissues -reaction catalyzed by this enzyme can utilize either NAD+ or NADP+ and is reversible -in extra hepatic tissues, which cannot perform urea cycle, glutamate dehydrogenase is then used to fix excess ammonia as glutamate >>this is then transported to the liver as glutamine and alanine

First step in amino acid degradation

-removal of nitrogen

Transamination reaction

-reversible -important in the production of non-essential AAs such as aspartate, glutamate, and alanine -aspartate and alanine can transaminate with a-ketoglutarate to form oxaloacetate and pyruvate respectively -when needed oxaloacetate and pyruvate can transaminate with glutamate to reform aspartate and alanine by the reverse reactions -AA can be made by the body if the corresponding a-keto acid derivative is available -for essential AAs, we lack the ability to synthesize the corresponding a-keto acids

Uses/Removal of NH4+

-some consumed in the biosynthesis of nitrogenous compounds such as nucleotides -amino groups are channeled into a single excretory end product -ost aquatic species are ammoniotelic, excreting amino nitrogen as ammonium -toxic ammonium is simply diluted in the surrounding water -birds and reptiles are uricoletic, excreting amino nitrogen as uric acid -most terrestrial vertebrates convert is to urea and excrete it - called ureotelic

Alanine cycle step 3

-the liver takes up alanine (from muscle that's in the blood stream) and converts it back to pyruvate by transamination with a-ketoglutarate

Alanine cycle step 2

-the nitrogen (in muscle glutamate) is then transferred from glutamate to pyruvate to form alanine, which is then released into the blood

Benzoate and phenyl butyrate are used to treat patients with CPS I and OTC defects

-these individuals will have high levels of the AAs glycine and glutamine in the blood >>given oral administration of benzoate and phenyl butyrate -benzoate converted into benzoyl CoA >>benzoyl CoA can conjugate with glycine to form hippuric acid which is then excreted -Phenylbutyrate is converted to phenylacetyl CoA by B-oxidation >>Rhenylacetyl CoA can then conjugate with glutamine to form phenylacetylglutamine which is also excreted -this therapeutic approach works b/c the conjugated amino acids are excreted >>the removal of these AAs then requires the body to resynthesizes more of them utilizing nitrogen molecules

Urea Cycle is linked to certain TCA cycle intermediates

-transamination of oxaloacetate by glutamate feeds nitrogen into the urea cycle in the form of aspartate -fumarate formed by the action of argininosuccinase is first hydrated to form malate, a TCA cycle intermediate -malate is then oxidized to form oxaloacetate

Net reaction of urea synthesis

CO2 + NH4+ + 3 ATP + aspartate + 2 H2O --> urea + 2 ADP + 2 Pi + AMP + PPi + fumarate -three molecules of ATP directly used -Pyrophosphate is rapidly hydrolyzed, thus the synthesis of one urea molecule consumes an equivalent of four ATP molecules -two are used in the synthesis of carbamoyl phosphate and two in the synthesis of argininosuccinate -liver is primary location of urea cycle; if damaged by alcoholism, hepatitis B, or chlorinated hydrocarbons then extraordinary measures such as liver transplantation would be needed

Glutamine transports nitrogen from peripheral tissues to the liver (step 2)

glutamine synthetase catalyzes the synthesis of glutamine from glutamate and NH4+ in an ATP-dependent reaction