amino acid oxidation and the urea cycle -...

Amino Acid Oxidation and the Urea Cycle

Amino Acid Oxidation and the Urea Cycle

Amino Acids:Amino Acids:

• Final class of biomolecules whose oxidation contributes significantly to the generation of energy

• Final class of biomolecules whose oxidation contributes significantly to the generation of energy

• Undergo oxidation in three metabolic circumstances in animals:

• Undergo oxidation in three metabolic circumstances in animals:

During normal synthesis and degradation of proteinsDuring normal synthesis and degradation of proteins

When a diet is rich in protein; amino acids are not storedWhen a diet is rich in protein; amino acids are not stored

During starvation or in diabetes mellitusDuring starvation or in diabetes mellitus

Overview of Amino Acid Catabolism:Overview of Amino Acid Catabolism:Intracellular

proteinIntracellular

proteinDietaryproteinDietaryprotein

Amino AcidsAmino Acids

NH4

+NH4

+

ExcretionExcretion

Biosynthesis of amino acidsnucleotides, biological amines

Biosynthesis of amino acidsnucleotides, biological amines

α-keto acidα-keto acid

TCA CycleTCA

Cycle

RespirationRespiration

GlucoseGlucose

Metabolic Fates of Amino GroupsMetabolic Fates of Amino Groups

Most amino acids are metabolized in the liver

Most amino acids are metabolized in the liver

• Some of the ammonia generated is used in biosynthesis; the excess is excreted

• Some of the ammonia generated is used in biosynthesis; the excess is excreted

• Excess ammonia generated in extrahepatic tissues is transported to liver for conversion to the appropriate excreted form

• Excess ammonia generated in extrahepatic tissues is transported to liver for conversion to the appropriate excreted form

Removal of the α-amino groups occurs in the cytosol by transamination reactions

catalyzed by aminotransferases (transaminases):

Removal of the α-amino groups occurs in the cytosol by transamination reactions

catalyzed by aminotransferases (transaminases):

α-keto acid2 + amino acid

1α-keto acid

2 + amino acid

1

α-keto acid1 + amino acid

2α-keto acid

1 + amino acid

2

In liver α-keto acid1 is usually α-Kg; in muscle

it is usually pyruvateIn liver α-keto acid

1 is usually α-Kg; in muscle

it is usually pyruvate

COO-COO-

C=OC=O

CH2CH2

COO-COO-

CH2CH2

α-Kgα-Kg

++COO-COO-

H3N+H3N+

RR

CC HH

amino acidamino acid

COO-COO-

C-HC-H

CH2CH2

COO-COO-

CH2CH2

NH3+NH3+ COO-COO-

RR

C=OC=O++

glutamateglutamateα-keto acidα-keto acid

COO-COO-

C=OC=O

CH2CH2

COO-COO-

CH2CH2

α-Kgα-Kg

COO-COO-

H3N+H3N+

CH3CH3

CC HH++

alaninealanine

GPTGPT

alanine

amino-

transferase

alanine

amino-

transferase

COO-COO-

C-HC-H

CH2CH2

COO-COO-

CH2CH2

NH3+NH3+

glutamateglutamate

++

COO-COO-

CH3CH3

C=OC=O

pyruvatepyruvate

COO-COO-

C=OC=O

CH2CH2

COO-COO-

CH2CH2

α-Kgα-Kg

COO-COO-

C-HC-H

CH2CH2

COO-COO-

CH2CH2

NH3+NH3+

glutamateglutamate

++

OAAOAA

++

aspartateaspartate

COO-COO-

H3N+H3N+

CH2CH2

CC HH

COO-COO-

C=OC=OCOO-COO-

CH2CH2

COO-COO-

Aspartate aminotransferase or Glutamate-OAA transaminase (GOT)Aspartate aminotransferase or Glutamate-OAA transaminase (GOT)

Serum GPT (SGPT) and GOT (SGOT) are sensitive indicators for a number of disease

conditions.

Serum GPT (SGPT) and GOT (SGOT) are sensitive indicators for a number of disease

conditions.

• During heart attacks, damaged heart cells leak aminotransferases.

• During heart attacks, damaged heart cells leak aminotransferases.

• Damaged liver cells also leak aminotransferases. SGPT and SGOT levels are monitored in people exposed to industrial chemicals.

• Damaged liver cells also leak aminotransferases. SGPT and SGOT levels are monitored in people exposed to industrial chemicals.

The effect of transamination is to collect amino groups from many amino acids and

convert them into one, glutamate

The effect of transamination is to collect amino groups from many amino acids and

convert them into one, glutamate

• Glutamate channels amino groups into biosynthetic pathways or into reactions where nitrogenous waste products are

formed

• Glutamate channels amino groups into biosynthetic pathways or into reactions where nitrogenous waste products are

formed

How are amino groups removed from glutamate and prepared for excretion?How are amino groups removed from glutamate and prepared for excretion?

Glutamate is transported into the mitochondrial matrix where it undergoes oxidative deamination catalyzed by glutamate d’hase:

Glutamate is transported into the mitochondrial matrix where it undergoes oxidative deamination catalyzed by glutamate d’hase:

GlutamateGlutamate

NAD(P)+NAD(P)+

NAD(P)HNAD(P)H

glutamate d’haseglutamate d’haseα-Kg + NH4α-Kg + NH4

Ammonia is extremely toxic to animal tissues; it is converted to glutamine for

transport from extrahepatic tissues to the liver or kidneys.

Ammonia is extremely toxic to animal tissues; it is converted to glutamine for

transport from extrahepatic tissues to the liver or kidneys.

glutamateglutamate

COO-COO-

C-HC-H

CH2CH2

COO-COO-

CH2CH2

NH3+NH3+

NH4NH4

glutamine synthetaseglutamine synthetaseCOO-COO-

C-HC-H

CH2CH2

C=OC=O

CH2CH2

NH3+NH3+

H2NH2N

glutamineglutamine

In the liver glutamine is converted back to glutamate by glutaminase:

In the liver glutamine is converted back to glutamate by glutaminase:

COO-COO-

C-HC-H

CH2CH2

C=OC=O

CH2CH2

NH3+NH3+

H2NH2N

glutamineglutamine

NH4+NH4+

glutaminaseglutaminase

H2OH2O

UREAUREA

glutamateglutamate

COO-COO-

C-HC-H

CH2CH2

COO-COO-

CH2CH2

NH3+NH3+

• Glutamine is the major transport form of ammonia; it is present in blood in much higher concentrations than other amino acids.

• Glutamine is the major transport form of ammonia; it is present in blood in much higher concentrations than other amino acids.

• Alanine also plays a role in transport of amino groups to the liver by the glucose-alanine cycle:

• Alanine also plays a role in transport of amino groups to the liver by the glucose-alanine cycle:

AA glutamate

Muscle

glutamate

alaninepyr

+ α−Kg

Liver

alanine

α−Kg

glutamatepyr +

glucose

Habitat determines the Molecular Pathway for Nitrogen Excretion

Habitat determines the Molecular Pathway for Nitrogen Excretion

• Aquatic organisms (bacteria, protozoa, fish) release ammonia to their aqueous enviroment (ammonotelic)

• Aquatic organisms (bacteria, protozoa, fish) release ammonia to their aqueous enviroment (ammonotelic)

• Birds and reptiles convert amino nitrogen into uric acid; they cannot carry enough water for the excretion of

nitrogen as urea (uricotelic)

• Birds and reptiles convert amino nitrogen into uric acid; they cannot carry enough water for the excretion of

nitrogen as urea (uricotelic)

• Terrestrial animals excrete amino nitrogen in the form of urea (ureotelic)

• Terrestrial animals excrete amino nitrogen in the form of urea (ureotelic)

• Plants recycle virtually all amino groups; there is no general pathway for nitrogen excretion

• Plants recycle virtually all amino groups; there is no general pathway for nitrogen excretion

The Urea CycleThe Urea Cycle

• Discovered by Hans Krebs and Kurt Hanseleit

• Discovered by Hans Krebs and Kurt Hanseleit

• Occurs in the liver• Occurs in the liver

• Takes place in two intracellular compartments; the cytosol and the mitochondrial matrix

• Takes place in two intracellular compartments; the cytosol and the mitochondrial matrix

NH4+NH4+ HCO3

-HCO3-++ carbamoyl phosphate

synthetase I

carbamoyl phosphate

synthetase I

2 ATP2 ATP2 ADP,

Pi2 ADP,

Pi

H2N-C-O-P-O-H2N-C-O-P-O-

OOOO

O-O-

carbamoyl phosphatecarbamoyl phosphate

ornithine transcarbamoylase

ornithine transcarbamoylase

NH3-(CH2)3-CH-COO-NH3-(CH2)3-CH-COO-

NH3+NH3+

OrnithineOrnithineH2N-C-NH-(CH2)3-CH-COO-H2N-C-NH-(CH2)3-CH-COO-

NH3+NH3+

CitrullineCitrulline

OO

PiPi

Transported to cytosolTransported to cytosol

H2N-C-NH-(CH2)3-CH-COO-H2N-C-NH-(CH2)3-CH-COO-

NH3+NH3+

CitrullineCitrulline

OO AspartateAspartate

ATPATP

PPiPPi

arginosuccinate

synthetase

arginosuccinate

synthetase

-C-NH-(CH2)3-CH-COO--C-NH-(CH2)3-CH-COO-

NH3+NH3+NH2

+NH2+

-OOC-CH2-CH-NH-OOC-CH2-CH-NHCOO-COO-

ArginosuccinateArginosuccinate

-C-NH-(CH2)3-CH-COO--C-NH-(CH2)3-CH-COO-

NH3+NH3+NH2

+NH2+

-OOC-CH2-CH-NH-OOC-CH2-CH-NHCOO-COO-

ArginosuccinateArginosuccinate

arginosuccinate lyasearginosuccinate lyase

-OOC-CH=CH-COO--OOC-CH=CH-COO-

FumarateFumarate

-C-NH-(CH2)3-CH-COO--C-NH-(CH2)3-CH-COO-

NH3+NH3+NH2

+NH2+

NH2NH2

ArginineArginine

-C-NH-(CH2)3-CH-COO--C-NH-(CH2)3-CH-COO-

NH3+NH3+NH2

+NH2+

NH2NH2

ArginineArginine

arginasearginase

NH3-(CH2)3-CH-COO-NH3-(CH2)3-CH-COO-

NH3+NH3+

OrnithineOrnithine

H2N-C-NH

2H

2N-C-NH

2

OO

UREAUREA

Back to mitochondrionBack to mitochondrion

Overall equation for Urea synthesis:Overall equation for Urea synthesis:

NH3 + HCO3- + Aspartate + 3 ATPNH3 + HCO3- + Aspartate + 3 ATP

urea + fumarate + 2ADP + 2Pi + AMP + PPiurea + fumarate + 2ADP + 2Pi + AMP + PPi

Regulation of the Urea Cycle:Regulation of the Urea Cycle:

• Carbamoyl Phosphate Synthetase I is allosterically activated by

N-acetylglutamate.

• Carbamoyl Phosphate Synthetase I is allosterically activated by

N-acetylglutamate.

• High levels of transamination during amino acid breakdown lead to elevated glutamate with concommitant increases in the concentration of N-acetylglutamate .

• High levels of transamination during amino acid breakdown lead to elevated glutamate with concommitant increases in the concentration of N-acetylglutamate .

Breakdown of Individual Amino AcidsBreakdown of Individual Amino Acids

Degradation of the carbon skeletons of the 20 common amino acids yields one of 7

intermediates: a-Kg, succinyl CoA, pyruvate, fumarate, OAA, acetoacetate, acetyl CoA

Degradation of the carbon skeletons of the 20 common amino acids yields one of 7

intermediates: a-Kg, succinyl CoA, pyruvate, fumarate, OAA, acetoacetate, acetyl CoA

α-Kg, succinyl CoA, pyruvate, fumarate, OAA can all serve as precursors for glucose synthesis; hence amino acids giving rise to

these intermediates are Glucogenic.

α-Kg, succinyl CoA, pyruvate, fumarate, OAA can all serve as precursors for glucose synthesis; hence amino acids giving rise to

these intermediates are Glucogenic.

Acetoacetate and acetyl CoA can serve as precursors for fatty acid or ketone

synthesis; hence amino acids giving rise to these compounds are termed Ketogenic

Acetoacetate and acetyl CoA can serve as precursors for fatty acid or ketone

synthesis; hence amino acids giving rise to these compounds are termed Ketogenic

Alanine, Serine, Glycine,

Cysteine, Threonine,

Tryptophan

Alanine, Serine, Glycine,

Cysteine, Threonine,

Tryptophan PyruvatePyruvate

Isoleucine, Leucine,

Threonine, Tryptophan

Isoleucine, Leucine,

Threonine, Tryptophan Acetyl CoAAcetyl CoA

Leucine, Lysine,

Phenylalanine,

Tyrosine

Leucine, Lysine,

Phenylalanine,

Tyrosine

AcetoacetateAcetoacetate

Arginine, Glutamine,

Glutamate, Proline, Histidine

Arginine, Glutamine,

Glutamate, Proline, Histidine α−Kgα−Kg

Isoleucine, Methionine,

Valine

Isoleucine, Methionine,

ValineSuccinyl CoASuccinyl CoA

Aspartate, Tyrosine,

Phenylalanine

Aspartate, Tyrosine,

PhenylalanineFumarateFumarate

Aspartate,

Asparagine

Aspartate,

AsparagineOAAOAA

ThreonineThreonineH3C-C-C-COO-H3C-C-C-COO-

HH

NH2NH2HOHO

HH

C-COO-C-COO-

NH2NH2

HHHH

GlycineGlycine

C-COO-C-COO-

NH2NH2

HOCH2HOCH2

HH

SerineSerineCH3-C-COO-CH3-C-COO-

OO

PyruvatePyruvate

TryptophanTryptophan

C-COO-C-COO-

NH2NH2

CH2CH2

HH

NHNH

C-COO-C-COO-

NH2NH2

CH3CH3

HHAlanineAlanine

C-COO-C-COO-

NH2NH2

HS-CH2HS-CH2

HH

CysteineCysteine

• Alanine is converted to pyruvate by transamination.• Alanine is converted to pyruvate by transamination.

• Asparagine is converted to Aspartate by Asparaginase:• Asparagine is converted to Aspartate by Asparaginase:

C-COO-C-COO-

NH2NH2

C-CH2C-CH2

HHOO

NH2NH2

C-COO-C-COO-

NH2NH2

C-CH2C-CH2

HHOO

-O-O

• Aspartate can be converted to OAA by transamination:• Aspartate can be converted to OAA by transamination:

Aspartate + α-KG Glutamate + OAAAspartate + α-KG Glutamate + OAA

• Aspartate degradation via the urea cycle yields fumarate.• Aspartate degradation via the urea cycle yields fumarate.

Methionine, Valine, IsoleucineMethionine, Valine, Isoleucine

Propionyl CoAPropionyl CoA

CH3-CH2-C-S-CoACH3-CH2-C-S-CoAOO

-OOC-CH2-CH2-C-S-CoA-OOC-CH2-CH2-C-S-CoAOO

Succinyl CoASuccinyl CoA

Arginine, Glutamine, Histidine, Proline Arginine, Glutamine, Histidine, Proline

GlutamateGlutamate

Glutamate D’haseGlutamate D’hase

α−Kgα−Kg

Leucine and Lysine are the only two purely ketogenic amino acids. HMG-CoA is an

intermediate in leucine degradation.

Leucine and Lysine are the only two purely ketogenic amino acids. HMG-CoA is an

intermediate in leucine degradation.

• Initial steps in valine, leucine, isoleucine degradation are identical:

• Initial steps in valine, leucine, isoleucine degradation are identical:

transamination to the corresponding a-keto acidtransamination to the corresponding a-keto aciddecarboxylation to the corresponding CoA derivativedecarboxylation to the corresponding CoA derivativedehydrogenation to form a double bonddehydrogenation to form a double bond

Defect in the decarboxylation reaction results in Maple Syrup Urine Disease, which is fatal unless treated early in life.

Defect in the decarboxylation reaction results in Maple Syrup Urine Disease, which is fatal unless treated early in life.

Phenylalanine is converted to Tyrosine by Phenylalanine Hydroxylase; then the pathway proceeds with the breakdown of tyrosine to fumarate plus acetoacetate. Homogentisate is an intermediate in this pathway.

Phenylalanine is converted to Tyrosine by Phenylalanine Hydroxylase; then the pathway proceeds with the breakdown of tyrosine to fumarate plus acetoacetate. Homogentisate is an intermediate in this pathway.

Alkaptonuria results in the urinary excretion of excess homogentisate; air oxidation causes this compound to turn dark. This disease is not fatal, individuals tend to suffer arthritis in later life.

Alkaptonuria results in the urinary excretion of excess homogentisate; air oxidation causes this compound to turn dark. This disease is not fatal, individuals tend to suffer arthritis in later life.

OHOH

OHOH-OOC-H

2C-OOC-H

2C homogentisatehomogentisate

Phenylketonuria results from absence of phenylalanine hydroxylase. Phe is converted to phenylpyruvate and excreted. Severe mental retardation occurs unless infants are immediately placed on a diet low in Phe.

Phenylketonuria results from absence of phenylalanine hydroxylase. Phe is converted to phenylpyruvate and excreted. Severe mental retardation occurs unless infants are immediately placed on a diet low in Phe.

CH2

CH2

C=OC=O

COO-COO-

phenylpyruvatephenylpyruvate

Human Genetic Disorders of Amino Acid Catabolism

Human Genetic Disorders of Amino Acid Catabolism

ConditionConditionIncidenceIncidence

(per 100,000 births)(per 100,000 births) Defective EnzymeDefective Enzyme SymptomsSymptoms

AlkaptonuriaAlkaptonuria 0.40.4 Homogentisate DioxygenaseHomogentisate Dioxygenase

Dark pigment in urine; arthritis in late lifeDark pigment in urine; arthritis in late life

Maple Syrup Urine DiseaseMaple Syrup Urine Disease

0.40.4 Branched chain α-keto acid dedydrogenaseBranched chain α-keto acid dedydrogenase

Mental retardation; convulsions; early death

Mental retardation; convulsions; early death

PhenylketonuriaPhenylketonuria 88 phenylalanine hydroxylasephenylalanine hydroxylase

Neonatal vomiting; mental retardationNeonatal vomiting; mental retardation

Methylmalonic Acidemia (MMA)Methylmalonic Acidemia (MMA)

(<0.5)(<0.5) Methylmalonyl CoA Mutase Methylmalonyl CoA Mutase

Mental retardation; convulsions; early death

Mental retardation; convulsions; early death