Amono Acid Metabolism

Chapter 17 ---2: Amino Acid Oxidation and the Production of Urea

Nitrogen Excretion and the Urea Cycle 

the ammonia in the mitochondria of hepatocytes is converted to urea via the urea cycle. This pathway was discovered in 1932 by Hans Krebs and a medical student associate, Kurt Henseleit. Urea production occurs almost exclusively in the liver.

Urea Is Formed in the Liver 

Using thin slices of liver suspended in a buffered aerobic medium, Krebs and Henseleit found that the rate of urea formation from ammonia was greatly accelerated by adding any one of three αamino acids: ornithine, citrulline, or arginine.

ornithine and citrnlline can serve as successive precursors of arginine. Note that citrulline and ornithine are nonstandard amino acids that are not found in proteins.

The Production of Urea from Ammonia Involves Five Enzymatic Steps 



The urea cycle begins inside the mitochondria of hepatocytes, but three of the steps occur in the cytosol; the cycle thus spans two cellular compartments . The first amino group to enter the urea cycle is derived from ammonia(Deamination) .together with HCO3- produced by mitochondrial respiration, to form carbamoyl phosphate in the matrix

HCO3− ATP ADP

Carbamoyl Phosphate Synthase (Type I) catalyzes a 3-step reaction, with HO carbonyl phosphate and NH3 carbamate intermediates. Pi Ammonia is the N input. H2N

O C

OPO32−

carbonyl phosphate

O C

The reaction, which ATP involves cleavage of 2 ~P bonds of ATP, is essentially ADP irreversible.

O

H2N

C

O−

carbamate

OPO32−

carbamoyl phosphate

carbamoyl phosphate synthetase I and II 

carbamoyl phosphate synthetase I. The mitochondrial form of the enzyme is distinct from the cytosolic (II) form, which has a separate function in pyrimidine biosynthesis (Chapter 21). Carbamoyl phosphate synthetase I is a regulatory enzyme; it requires N-acetylglutamate as a positive modulator (see below). Carbamoyl phosphate may be regarded as an activated carbamoyl group donor.

H H3N+

C

O

H

N-ac glutamat COO−

CH2

H3C

C

N H

C

COO−

CH2

CH2

CH2

COO−

COO−

Carbamoyl Phosphate Synthase has an absolute requirement for an allosteric activator N-acetylglutamate. This derivative of glutamate is synthesized from acetylCoA & glutamate when cellular [glutamate] is high, signaling an excess of free amino acids due to protein breakdown or dietary intake.

urea cycle 

The carbamoyl phosphate now enters the urea cycle, which entails four enzymatic steps. Carbamoyl phosphate donates its carbamoyl group to ornithine to form citrulline and release Pi in a reaction catalyzed by ornithine transcarbamoylase. The citrulline is released from the mitochondrion into the cytosol.

cytosol mitochondrial matrix carbamoyl phosphate Pi ornithine ornithine urea arginine

citrulline citrulline aspartate argininosuccinate

fumarate For each cycle, citrulline must leave the mitochondria, and ornithine must enter the mitochondrial matrix. An ornithine/citrulline transporter in the inner mitochondrial membrane facilitates transmembrane fluxes of citrulline & ornithine.

mitochondrial matrix and the cytosol.

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O H 2N

C

NH2

urea Most terrestrial land animals convert excess nitrogen to urea, prior to excreting it. Urea is less toxic than ammonia. The Urea Cycle occurs mainly in liver. The 2 nitrogen atoms of urea enter the Urea Cycle as NH3 (produced mainly via Glutamate Dehydrogenase) and as the amino N of aspartate. The NH3 and HCO3− (carbonyl C) that will be part of urea are incorporated first into carbamoyl phosphate.

The Citric Acid and Urea Cycles Are Linked 

The fumarate produced in the argininosuccinate lyase reaction is also an intermediate of the citric acid cycle. Fumarate enters the mitochondria, where the combined activities of fumarase and malate dehydrogenase transform fumarate into oxaloacetate . Because the reactions of the urea and citric acid cycles are inextricably intertwined, together they have been called the "Krebs bicycle."

Fumarate produced in the cytosol by argininosuccinate lyase of the urea cycle enters the citric acid cycle in the mitochondrion and is converted in several steps to oxaloacetate. Oxaloacetate accepts an amino group from glutamate by transamination, and the aspartate thus formed leaves the mitochondrion and donates its amino group to the urea cycle in the argininosuccinate synthetase reaction.

COO−

COO−

COO−

CH2

COO−

CH2

CH2

CH2

CH2

CH2

HC

NH3+

COO−

+

C

O

COO−

C

O

COO−

+

HC

NH3+

COO−

aspartate α-ketoglutarate oxaloacetate glutamate

Aminotransferase (Transaminase) Fumarate is converted to oxaloacetate via Krebs Cycle enzymes Fumarase & Malate Dehydrogenase. Oxaloacetate is converted to aspartate via transamination (e.g., from glutamate). Aspartate then reenters Urea Cycle, carrying an amino group derived from another amino acid.

The Activity of the Urea Cycle Is Regulated 

The flux of nitrogen through the urea cycle varies with the composition of the diet. When the diet is primarily protein, the use of the carbon skeletons of amino acids for fuel results in the production of much urea from the excess amino groups. During severe starvation, when breakdown of muscle protein supplies much of the metabolic fuel, urea production also increases substantially, for the same reason.

The reaction catalyzed by carbamoyl phosphate synthetase I. The formation of carbamoyl phosphate in the mitochondrial matrix is strongly stimulated by the allosteric effector Nacetylglutamate . Note that the terminal phosphate groups of two molecules of ATP are used to form one molecule of carbamoyl phosphate: two activation steps occur in the carbamoyl phosphate synthetase I reaction.

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Figure 17-14 Synthesis of Nacetylglutamate, the allosteric activator of carbamoyl phosphate synthetase I, is stimulated by high concentrations of arginine. Increasing arginine levels signal the need for more flux through the urea cycle.

long term regulation 

These changes in demand for urea cycle activity are met in the long term by regulation of the rates of synthesis of the urea cycle enzymes and carbamoyl phosphate synthetase I in the liver. All five enzymes are synthesized at higher rates during starvation or in animals on very highprotein diets than in well-fed animals on diets containing primarily

The Urea Cycle Is Energetically Expensive 

The urea cycle brings together two amino groups and HCO3- to form a molecule of urea, which diffuses from the liver into the bloodstream, thence to be excreted into the urine by the kidneys. The overall equation of the urea cycle is



2NH4+ + HCO3- + 3ATP4- + H2O + 2ADP3- + 4Pi2- + AMP2- + 5H+

urea

3 ATP 

The synthesis of one molecule of urea requires four high-energy phosphate groups. Two ATPs are required to make carbamoyl phosphate, and one ATP is required to make argininosuccinate. In the latter reaction, however, the ATP undergoes a pyrophosphate cleavage to AMP and pyrophosphate, which may be hydrolyzed to yield two Pi.



It has been estimated that, because of the necessity of excreting nitrogen as urea instead of ammonia, ureotelic animals lose about 15% of the energy of the amino acids from which the urea was derived.

Hereditary deficiency of any of the Urea Cycle enzymes leads to hyperammonemia - elevated [ammonia] in blood. Total lack of any Urea Cycle enzyme is lethal. Elevated ammonia is toxic, especially to the brain. If not treated immediately after birth, severe mental retardation results.

Postulated mechanisms for toxicity of high [ammonia]: 1. High [NH3] would drive Glutamine Synthase: glutamate + ATP + NH3  glutamine + ADP + Pi This would deplete glutamate – a neurotransmitter & precursor for synthesis of the neurotransmitter GABA. 2. Depletion of glutamate & high ammonia level would drive Glutamate Dehydrogenase reaction to reverse: glutamate + NAD(P)+ α -ketoglutarate + NAD(P)H + NH4+ The resulting depletion of α -ketoglutarate, an essential Krebs Cycle intermediate, could impair energy metabolism in the brain.

Treatment of deficiency of Urea Cycle enzymes (depends on which enzyme is deficient): 

limiting protein intake to the amount barely adequate to supply amino acids for growth, while adding to the diet the α -keto acid analogs of essential amino acids.



Liver transplantation has also been used, since liver is the organ that carries out Urea Cycle.

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Table Nonessential and essential amino acids for humans and the albino rat Nonessential Essential Alanine Arginine* Asparagine Histidine Aspartate Isoleucine Cysteine Leucine Glutamate Lysine Glutamine Methionine Glycine Phenylalanine Proline Threonine Serine Tryptophan Tyrosine Valine * Essential in young, growing animals but not in adulta.

Summary 

Ammonia is highly toxic to animal tissues. Ureotelic animals (adult terrestrial amphibians and all mammals) excrete amino nitrogen as urea, formed in the liver by the urea cycle. Arginine is the immediate precursor of urea. Arginase hydrolyzes arginine to yield urea and ornithine, and arginine is resynthesized in the urea cycle.

Summary 

Ornithine is converted to citrulline at the expense of carbamoyl phosphate, and an amino group is transferred to citrulline from aspartate, re-forming arginine. Ornithine is regenerated in each turn of the cycle.

Summary 



 

Several of the intermediates and byproducts of the urea cycle are also intermediates in the citric acid cycle, and the two cycles are thus interconnected. The activity of the urea cycle is regulated at the levels of enzyme synthesis and allosteric regulation of the enzyme that forms carbamoyl phosphate. The formation of the nontoxic urea and of solid uric acid has a high ATP cost. Genetic defects in enzymes of the urea cycle can be compensated for by dietary regulation.

Overview of Amino Acid Catabolism: organ Relationships

Detoxification of Ammonia by the Liver: the Urea Cycle 

Amino acid N flowing to liver as:  Alanine

& glutamine  Other amino acids  Ammonia (from portal blood) 

Urea  chief

N-excretory compound

Flow of Nitrogen from Amino Acids to Urea in Liver 

Amino acid flow from muscle to liver 



Alanine & glutamine

Liver 

Transfers N to GLU  



GLN’ase Transaminases

Transfers GLU-N to: 

ASP 



NH3 



AST  Transamination route GDH  Trans-deamination route

Transfers N to urea

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28.2 Amino Acid Metabolism: An Overview The amino acid pool, the entire collection of free amino acids throughout the body, occupies a central position in amino acid metabolism.

1. Protein Degradation Dietary proteins are a vital source of amino acids. Discarded cellular proteins are another source of amino acids.

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1.2 Cellular Protein Degradation 

Cellular proteins are degraded at different rates. 

Ornithine decarboxylase has a half-life of 11 minutes.



Hemoglobin lasts as long as a red blood cell.



Υ-Crystallin (eye lens protein) lasts as long as the organism does. 41

Turnover 



Amino acids used for synthesizing proteins are obtained by degrading other proteins 

Proteins destined for degradation are labeled with ubiquitin.



Polyubiquinated proteins are degraded by proteosomes.

Amino acids are also a source of nitrogen for other biomolecules.

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Protein Turnover Is Tightly Regulated 

The turnover of cellular proteins is a regulated process requiring complex enzyme systems. Proteins to be degraded are conjugated with ubiquitin, a small conserved protein, in a reaction driven by ATP hydrolysis. A large, barrel-shaped complex called the proteasome digests the ubiquitinated proteins. The proteasome also requires ATP hydrolysis to function.

Ubiquitin: Proteins are usually tagged for selective destruction in proteolytic complexes called proteasomes by covalent attachment of ubiquitin, a small, compact, highly conserved protein.

ubiquitin

PDB 1TBE

However, some proteins may be degraded by proteasomes without ubiquitination. An isopeptide bond links the terminal carboxyl of ubiquitin to the ε -amino group of a lysine residue of a "condemned" protein.

H H3N+

C CH2 CH2 CH2 CH2 + NH 3

COO−

The joining of ubiquitin to a condemned protein is ATP-dependent. Three enzymes are involved, designated E1, E2 & E3. The ubiquitin pathway is a multi-step process in which an ubiquitin molecule is activated by an E1 enzyme, transferred to an E2 enzyme, and then covalently attached to the protein substrate either directly or in conjunction with E3 enzyme . Proteins destined to be degraded are subject to multiple rounds of ubiquitin attachment and are then proteolyzed by the 26S proteasome.

O ubiquitin

C

S

Cys

E2

+

H2N

Lys

protein to be degraded

E3 (Ubiquitin-Protein Ligase) O ubiquitin

C

N

Lys

protein to be degraded

+ HS

Cys

E2

H

A Ubiquitin-Protein Ligase (E3) then transfers activated ubiquitin to the ε -amino group of a Lys residue of a protein recognized by that E3, forming an isopeptide bond.

destruction Primary structure of a protein targeted for degradation box COO−

H2 N

chain of ubiquitins

More ubiquitins are added to form a chain of ubiquitins. The terminal carboxyl of each ubiquitin is linked to the ε amino group of a Lys residue of the adjacent ubiquitin. A chain of 4 or more ubiquitins (linked via Lys29 or Lys48) targets proteins for degradation in proteasomes.

destruction Primary structure of a protein targeted for degradation box COO−

H2 N

chain of ubiquitins

Some proteins (e.g., mitotic cyclins involved in cell cycle regulation) have a destruction box sequence recognized by a domain of the corresponding Ubiquitin Ligase.

Proteasomes: Selective protein degradation occurs in the proteasome, a large protein complex in the nucleus & cytosol of eukaryotic cells.

α

20 S Proteasome (yeast) closed state

β β α two views

PDB 1JD2

The proteasome core complex, with a 20S sedimentation coefficient, contains 2 each of 14 different polypeptides.  7 α -type proteins form each of the two α rings, at the ends of the cylindrical structure.  7 β -type proteins form each of the 2 central β rings.

α

20 S Proteasome (yeast) closed state

β β α two views

PDB 1JD2

The 20S proteasome core complex encloses a cavity with 3 compartments joined by narrow passageways. Protease activities are associated with 3 of the β subunits, each having different substrate specificity.

1.

One catalytic β -subunit has a chymotrypsin-like activity with preference for tyrosine or phenylalanine at the P1 (peptide carbonyl) position.

2.

One has a trypsin-like activity with preference for arginine or lysine at the P1 position.

3.

One has a post-glutamyl activity with preference for glutamate or other acidic residue at the P1 position.

Proteasome evolution: Proteasomes are considered very old. They are in archaebacteria, but not most eubacteria, although eubacteria have alternative proteindegrading complexes. 

The archaebacterial proteasome has just 2 proteins, α & β , with 14 copies of each.



The eukaryotic proteasome has evolved 14 distinct proteins that occupy unique positions within the proteasome (7 α -type & 7 β -type).