Amino acid degradation

The reaction mechanism of aminotransferase

1. The amino acid's nucleophilic α-amino group attacks the enzyme-PLP Schiff base carbon atom to form an amino acid-PLP Schiff base. 2,3 The amino acid-PLP Schiff base tautomerizes to an α-keto acid-PMP Schiff base by the removal of the amino acid α hydrogen and protonation of the Schiff base carbon atom. 4. The α-keto acid-PMP Schiff base is hydrolyzed to PMP and an α-keto acid. 5. To complete the reaction, a second α-keto acid replaces the one that is released, and this is converted to an amino acid in a reversal of the reaction steps. Removal of the amino acid α hydrogen produces a resonance-stabilized carbanion whose electrons are delocalized all the way to the coenzyme's pyridinium nitrogen atom; that is, PLP functions as an electron sink.

Six amino acids are degraded to pyruvate

All or part of the carbon skeletons of six amino acids (alanine, serine, cysteine, glycine, threonine, and tryptophan) can be converted to pyruvate (three-carbon). • Alanine, serine, and cysteine (three-carbon) are converted in whole. • Alanine: via a simple transamination reaction. • Serine: via a deamination by serine dehydratase. • Cysteine: the sulfhydryl group is eliminated first, followed by a transamination reaction. • One of the break down products of tryptophan is alanine.

Animals excrete nitrogen waste in several different forms

Amino acids are nitrogen-containing molecules, whose degradation involves how to safely dispose the toxic nitrogen waste. • In plants, almost all nitrogen are absorbed and re-enter the biosynthetic pathway. • In animals, nitrogen wastes are excreted in one of the three following forms. • Ammonia: toxic but simple; excreted by most marine animals (can be diluted easily through the environment). • Urea: very soluble; excreted by many land animals including mammals. • Uric acid: insoluble (excretion as paste allows the animals to conserve water); excreted by birds and reptiles.

Two amino acids are degraded to oxaloacetate

Aspartate and asparagine (four-carbon) are converted into oxaloacetate (four-carbon). • Aspartate: a simple transamination reaction yields oxaloacetate. • can also be converted to fumarate in the urea cycle. • Asparagine: first hydrolyzed by L-asparaginase to yield NH4 + and aspartate.

Five amino acids are degraded to α-ketoglutarate

Degradation of five amino acids (glutamate, glutamine, proline, arginine, and histidine) leads to α-ketoglutarate (five-carbon). • Glutamate: through oxidative deamimation. • The other four amino acids are first converted to glutamate: • Glutamine: hydrolysis by glutaminase. • Arginine (six-carbon): first converted to ornithine (the urea cycle) and then to glutamate γ-semialdehyde, which is oxidized to glutamate. • Proline: also via glutamate γ-semialdehyde intermediate. • Histidine (six-carbon): via a set of four reaction steps. One carbon atom is transferred to THF.

Hydroxylation of phenylalanine is the first step in its degradation

Degradation of phenylalanine begins with hydroxylation of phenylalanine to form tyrosine, a reaction catalyzed by phenylalanine hydroxylase (also called phenylalanine-4-monooxygenase). • The enzyme requires a redox cofactor tetrahydrobiopterin, which passes electrons to O2 and becomes oxidized to dihydrobiopterin in the process. • Dihydrobiopterin is subsequently reduced to tetrahydrobiopterin by dihydrobiopterin reductase in a reaction that requires NAD(P)H.

Four amino acids are degraded to succinyl-CoA

Degradation of the carbon skeleton of methionine, isoleucine, threonine, and valine converge on propionyl-CoA (three-carbon), which is converted to succinyl-CoA. • Methionine (five-carbon): one carbon atom is transferred through Sadenosylmethionine, a methyl group carrier. One carbon atom is released as CO2. • Isoleucine (six-carbon): one carbon atom is released as CO2, the other two as acetyl-CoA. Valine (five-carbon): two carbon atoms are released as CO2. • Threonine (four-carbon): converges on α-ketobutyrate with methionine degradation. One carbon atom is released as CO2. This is the major degradation pathway in human.

Alanine transports ammonia from skeletal muscles to the liver

During intense exercise, muscle proteins are rapidly broken down to amino acids to be used as biological fuels. • Amino groups removed from these amino acids are first combined with pyruvate to form alanine in the muscle. • Alanine is transported through blood to the liver, where it undergoes transammination. • Pyruvate is used in the liver to synthesize glucose which is transported back to the muscle, completing the glucose-alanine cycle. • The cycle also helps eliminate pyruvate generated in the muscle from glycolysis during anaerobic exercise and prevents lactic acid build-up.

Degradation of aromatic amino acids requires molecular oxygen

For the aromatic amino acids, oxygenase is used to break the aromatic ring. Two types of oxygenase are used: • Monooxygenase or mixed-function oxygenase incorporates only one atom of O2 into the product (the other into H2O). It catalyzes a hydroxylation reaction. • Dioxygenase incorporates both atoms of O2 into the product. It is used to break aromatic rings in biological systems.

The urea and citric acid cycles are linked

Fumarate (produced in step 5 of the urea cycle) is also an intermediate of the citric acid cycle. Thus, these two cycles are interconnected (Krebs bicycle). • Fumarate can be converted to malate and then to oxaloacetate in the cytosol. • Oxaloacetate can be converted to glucose by gluconeogenesis. • Oxaloacetate can be transaminated to form aspartate, which serves as nitrogen donor in the urea cycle (aspartateargininosuccinate shunt).

Oxidative deamination of glutamate releases its α-amino group

Glutamate releases its amino group through oxidative deamination, catalyzed by glutamate dehydrogenase. • The enzyme can use either NAD+ or NADP+ as electron acceptor and the reactions occurs within mitochondrial matrix. • The overall deamination reaction for any amino acid is therefore a combination of transamination and oxidative deamination

Degradation of glycine and threonine

Glycine (two-carbon) • Pathway 1: separation of three central atoms via a complicated reaction mechanism • to yield CO2, NH4 + • and a methylene group in the form of N5, N10- methylene-tetrahydrofolate (THF). • Pathway 2: converted to serine utilizing N5, N10- methylene-THF to provide the necessary one-carbon unit. • Threonine (four-carbon) • Pathway 1: conversion to glycine and acetyl-CoA. • Pathway 2: conversion to succinyl-CoA (major pathway in human).

Overview of amino acid degradation in mammals

In mammals, all amino acids are broken down using the same general strategy: • the α-amino group is separated from the carbon skeleton in the form of ammonia which enters the urea cycle to be excreted. • the carbon skeleton (keto acid) enters the central metabolism pathway for oxidation or biosynthesis.

Glutamine is a non-toxic form of ammonia

In many tissues, free ammonia is generated and must be converted into a non-toxic form. • Glutamine is the most important non-toxic form of ammonia in the body. • Free ammonia is combined with glutamate to yield glutamine by the action of glutamine synthetase. • The reaction requires ATP hydrolysis. • Glutamine can be safely transported to the liver and and ammonia is released in mitochondria by glutaminase and subsequently disposed via the urea cycle. • Ammonia assimilated in glutamine can also serve as a source of nitrogen in many biosynthetic pathways (next lecture).

Overview of the urea cycle

In the urea cycle, ammonia is converted to urea and then excreted into the urine. • Urea cycle occurs almost exclusively in the liver. • One of the nitrogen atoms of urea is derived directly from free ammonia. • The source of the other nitrogen atom is aspartate, which is generated by transfer of α-amino acid from glutamate to oxaloacetate. • The carbon atom of urea comes from CO2 (in the form of HCO3 - ).

Strategies to separate α-amino groups from carbon skeletons

Major • Transamination: in the liver, α-amino groups from most amino acids are transferred to α-ketoglutarate to form glutamate and corresponding α-keto acids. • Deamination: glutamate undergoes oxidative deamination to serve as a nitrogen donor (in the form of ammonia) for the urea cycle. Other • In skeletal muscle, α-amino groups from certain amino acids are transferred to pyruvate to form alanine, which is shuttled to the liver for degradation. • The α-amino group in aspartate is also a nitrogen donor for the urea cycle.

Seven amino acids are degraded to acetyl-CoA

Part of the carbon skeletons of tryptophan, lysine, phenylalanine, threonine, leucine, isoleucine, and tyrosine can be converted to acetyl-CoA. • Some of the final steps in degradative pathways for leucine, lysine, and tryptophan resemble steps in βoxidation of fatty acids. • Leucine (six-carbon): converted to three acetyl-CoA. • Lysine (six-carbon): converted to two acetyl-CoA and two CO2. • Tryptophan (eleven-carbon): the most complex pathway. Three carbon atoms are converted to pyruvate, four to acetyl-CoA, and four to CO2. • Phenylalanine, tyrosine (nine-carbon): Four carbon atoms are converted to fumarate, four to acetyl-CoA, and one to CO2

Phenylketonuria results from defects in phenylalanine degradation

Phenylketonuria is a genetic disease when phenylalanine hydroxylase activity is missing or deficient. • Excess phenylalanine undergoes transamination to yield phenylpyruvate, both of which will show up in high concentration in the urine. • A buildup of phenylalanine and phenylpyruvate impairs neurological development leading to intellectual deficits. • Screening newborns for phenylketonuria can prevent mental retardation by limiting dietary intake of phenylalanine.

Aminotransferases use pyridoxal phosphate

Pyridoxal phosphate (PLP) is a vitamin B6 derivative. • PLP works as a cofactor primarily in reactions involved in the metabolism of molecules with amino groups. • The functionality of PLP lies in its ability to undergo reversible transformation between its aldehyde form (PLP, which can accept an amino group), and its aminated form (pyridoxamine phosphate or PMP, which can donate an amino group). • PLP is generally covalently bound to an active site lysine residue through a Schiff base linkage.

Regulation of the urea cycle occurs at two levels

Short-term regulation: • Carbamoyl phosphate synthetase I is allosterically activated by N-acetylglutamate. • N-acetylglutamate is formed by combining glutamate and acetylCoA, catalyzed by N-acetylglutamate synthase. • The enzyme is active when glutamate and acetyl-CoA concentrations are high Long-term regulation: • Expression level of urea cycle enzymes increases during • high-protein diet • starvation, when protein is being broken down for metabolic energy

Acquisition of the first urea nitrogen atom

Step 1: coupling of NH4 + with bicarbonate to form carbamoyl phosphate. • This is the committed step of the urea cycle. • This reaction, which occurs inside liver mitochondria, is catalyzed by a mitochondrial enzyme, carbamoyl phosphate synthetase I (CPS I). a. Activation of biocarbonate by ATP to form carboxyphosphate. b. Nucleophilic attack of NH3 on carboxyphosphate, displacing the phosphate to form carbamic acid and phosphate. c. Activation of carbamic acid by a second ATP to form carbamoyl phosphate.

Citrulline moves the urea cycle into the cytoplasm

Step 2-3: the majority of reactions within the urea cycle occur within the cytoplasm. • To move to the cytoplasm, carbamoyl phosphate condenses with ornithine to form citrulline, catalyzed by ornithine transcarbamoylase [step 2]. • Citrulline is exported from the mitochondria as ornithine is imported to the mitochondria [step 3].

Acquisition of the second urea nitrogen atom

Step 4: aspartate is the donor of the second urea nitrogen atom. • Citrulline condenses with aspartate to form arginosuccinate in a reaction catalyzed by arginosuccinate synthetase. • The reaction involves the formation of a citrullyil-AMP intermediate, which is subsequently displaced by the aspartate amino group. The reaction is driven by the hydrolysis of pyrophosphate.

Release of urea and regeneration of ornithine

Step 5-7: release of urea. • Argininosuccinase cleaves argininosuccinate to form fumarate and arginine [step 5]. • Arginase cleaves both nitrogen atoms added in the urea cycle from arginine to form ornithine and free urea [step 6]. • Ornithine is imported back to the mitochondria matrix [step 7] for another round of the urea cycle.

The carbon skeletons of amino acids enter the citric acid cycle

The carbon skeletons of the amino acids are metabolized to seven major metabolic intermediates: acetyl CoA, acetoacetyl CoA, pyruvate, α-ketoglutarate, succinyl CoA, fumarate, and oxaloacetate (all enter the citric acid cycle) • Amino acids metabolized to acetyl CoA and acetoacetyl CoA are called ketogenic amino acids because they can only form ketone bodies. • Amino acids degraded to the remaining major intermediates are called glucogenic amino acids because they can be used to synthesize glucose. • Only two amino acids, leucine and lysine, are solely ketogenic.

Branched-chain amino acids are not degraded in the liver

The liver lacks specific aminotransferases for three branched-chain amino acids (leucine, isoleucine, and valine). • Extrahepatic tissues contain an aminotransferase (using pyruvate as the amino group acceptor) that allows all three branched-chain amino acids to be degraded as metabolic fuels. • The branched-chain α-keto acid dehydrogenase complex catalyzes oxidative decaboxylation of all three resulting α-keto acids, in each case releasing CO2 and acyl CoA derivatives (mechanistically similar to pyruvate dehydrogenase complex). • A genetic deficiency in branched-chain α-keto acid dehydrogenase complex leads to maple syrup urine disease, which can result in death in early infancy

Amino acids as a source of metabolic energy

The primary role of amino acids in the cell is to serve as building blocks for protein synthesis. • Amino acids (its carbon skeleton) can also be used as a source of metabolic energy • Dietary amino acids that exceed body's protein synthesis needs or left-over amino acids from normal protein turnover • Amino acid can not be stored for future use • Proteins in the body (muscle proteins in particular) can be broken down when carbohydrates are scarce (starvation, diabetes). • Use of amino acids as a source of metabolic energy varies greatly by organisms • Carnivores: ~ 90% of energy needs are met by amino acids immediately after a meal. • Herbivores: a small fraction of energy needs are met by amino acids. • Microorganisms: scavenge amino acids from their environment for fuel when needed. • Plants: amino acids normally are not a source of metabolic energy but can be degraded to form other metabolites.

The transamination reaction

The transamination reaction is catalyzed by aminotransferases (transaminases). • Different aminotransferases are specific for different donor amino acids but most use α-ketoglutarate as the α-amino group acceptor (particularly in the liver). • alanine aminotransferase and aspartate aminotransferase are the most abundant aminotransferases in the liver. • The level of aspartate aminotransferase in the blood is used as a diagnostic tool for liver diseases. • The transamination reactions are freely reversible (ΔG' ≈ 0 kJ/mol). The direction of these reactions is determined by the relative concentrations of substrates and products involved.

Energy cost of the urea cycle

The urea cycle makes one urea molecule at the cost of four "high energy" phosphate bonds (even though only three ATP molecules are consumed) • Step 1: 2 ATP hydrolyzed to 2 ADP and 2 Pi • Step 4: 1 ATP hydrolyzed to 1 AMP and 1 PPi (followed by rapid PPi hydrolysis) HCO3 - + NH4 + + 3 ATP + aspartate + 2H2O ⟶ urea + 2 ADP + 4 Pi + AMP + fumarate