Prof. Dr. Onn Hashim

Department of Molecular Medicine, University of Malaya

Mechanisms in the disposal of nitrogen from amino acids Catabolism of the carbon skeletons of amino acids Gluconeogenesis Biosynthesis of non-essential amino acids Conversion of amino acids to specialised products

Devlin TM, ed. (2006) Biochemistry with clinical relevance (6th edition) Wiley-Liss, New York. Onn Hashim (2003) Metabolisme tubuh manusia (2nd edition), UM Press.

Outline

Topics to be covered include:

References

• how nitrogens from amino acids are converted into urea through

the process of transamination and deamination

• how the carbon skeletons of amino acids can be used to form

glucose and ketone bodies

• that defects in the amino acid metabolism may cause various

clinical conditions and diseases such as phenylketonuria,

hyperammonemia and maple syrup urine disease

• that glucose may be generated from non-carbohydrate

substances like lactate, glycerol and amino acids by

gluconeogenesis

• that amino acids are not only used for protein synthesis but are

also precursors to other non-protein products such as creatine,

glutathione and neurotransmitters

Lecture series

At the end of the lecture series, students should be able to understand:

Overview of Amino Acid Metabolism

Amino acid pool

Glucose

Synthesis of proteins &

specialised products

Endogenous &

dietary proteins

Glucose

Ketone bodies

Urea

NH4+

Human urine nitrogen components.

Final product % excreted N

Urea 86.0

Creatinine 4.5

Ammonium ions 2.8

Uric acid 1.7

Others 5.0

Amino Acid 1

Amino Acid 2 Keto Acid 1

Keto Acid 2

transaminase

Transamination Amino acid Keto acid

C3 alanine pyruvate

C4 aspartate oxaloacetate

C5 glutamate a-ketoglutarate

N

C

N

H

Lys

O-

CH3

H

H2CO

P

-O

O

-O

Arg+

hydrogen bonds

C N+

-helix dipole

C Asp

O

-O

HO Tyr

NH3+

COO-

H C R

Catabolism of Amino Acids

tautormerisation

Mechanism of Transamination

Reaction

N

CH

N

C

H

COO-

R

CH3

P OCH2

OH

NH2

N

C

CH3

P OCH2

OH

HN(CH2)4

NH2

N

CH2

CH3

P OCH2

OH

NH2

NH2

N

CH

N

C

H

COO-

R'

CH3

P OCH2

OH

H

C

NH2

COO-

R

O

C COO-

R

NH2

N

CH2

CH3

P OCH2

OH

N

R COO-

C

O

C COO-

R'

H

C

NH2

COO-

R'

tautormerisation

(aldimine) (aldimine)

(ketimine) (ketimine) NH2

N

CH

CH3

P OCH2

OH

N

R' COO-

C

2

Clinical Relevance of Transamination

Oxidative Deamination

Liver diseases: serum ALT ↑ ; AST ↑

Heart diseases: serum ALT = ; AST ↑

Coupling of Transamination & Deamination

Amino Acid

Glutamate Keto Acid

-Ketoglutarate

transaminase

NAD+

NADH + NH4+

GDH

C H+H3N

COO-

CH2)2

COO-

( (

C

COO-

CH2)2

COO-

ONH4

+ H2O

NAD+ NADH + H+

+ +

(-KG) (GLU) glutamate

dehydrogenase

Transamination & Deamination

Liver: 2NH4+ Urea

Brain: -KG Glutamate Glutamine

Non-oxidative Deamination

Serine Pyruvate + NH4+

dehydratase Threonine -Ketobutyrate + NH4

+

dehydratase

Histidine Urocanate + NH4+

lyase Cysteine Pyruvate + NH4

+ + H2S desulphurase

Detoxification & Transportation of Ammonia

Muscle: NH4+ Glutamate -KG

NH4+

Pyruvate Alanine

NH4+

NH4+ released are used to synthesise nitrogenous compounds

NH4+ are toxic to CNS

Excess NH4+ are excreted

Gln synthetase

Transamination & Deamination

2ATP + CO2 + NH4 H2N C OPO3

O

+ 2ADP + Pi

C

NH

CH2)3

NH2O

HC

COO-

NH3+

(

COO-

CH2

HC NH

COO-

CNH2

NH

CH2)3

HC NH3+

COO-

(

NH3+

HC

CH2)3

NH

COO-

CNH2H2N

(

(

NH3+

CH2)3

HC

COO-

NH3+

COO-

CH2

HC NH3+

COO-

COO-

C

CH

COO-

H

H2N C NH2

O

H2O

ADP + P i

ATP

Citrulline Ornithine

Argininosuccinate

Aspartate

Arginine

Urea

Fumarate

Mitosol

Cytosol

Argininase

CPS

Orn T

Asp T

Argsuccinase

+

+

2– +

The Urea Cycle

Formation of CAP is the

rate limiting step

Factors determining

reactions proceed toward

catabolism of Glu & urea

synthesis:

use of NH4+ by CPS

oxidation of -KG

hydrolysis of ATP

(also stimulates GDH

to produce NH4+)

Glutamate

-KG

NAD(P)+

NH4+ + CO2

GDH

NAD(P)H + H+

Carbamoyl phosphate

2ATP

2ADP + Pi

N-Acetyl-Glu

Acetyl-CoA

CPS

Regulation of the Urea Cycle

Citrulline

Ornithine Argininosuccinate

Aspartate

Arginiine Fumarate

Malate

OAA

Krebs Cycle

Amino acid

Keto acid Carbamoyl phosphate

Cytosol

Connection bet. Urea & Krebs Cycles

Citrulline Ornithine

Argininosuccinate

Aspartate

Arginine

Urea

Fumarate

Hyperammonemia type I (Carbamoyl phosphate synthetase)

2ATP + CO2 + NH4+

Carbamoyl phosphate + 2ADP + Pi

Hyperammonemia type II (Ornithine transcarbamoylase)

Citrullinuria (Argininosuccinate synthetase)

Argininosuccinate acidemia (Argininosuccinate lyase)

Hyperargininemia (Arginase)

Defects of the Urea Cycle

Treatment of Hyperammonemia Haemodialysis or Exchange Transfusion

Low protein diet

Dietary regulation – less but regular consumption

Treatment with Benzoate or Phenyl Acetate

• Benzoate + Glycine → Hippuric Acid

• Phenyl Acetate + Glutamine → Phenylacetylglutamine

Supplementation with Arginine or Citrulline

Blood Urea Nitrogen (BUN) Low ~ suspected liver problem

High ~ suspected kidney problem

Treatment

The carbon skeletons of twenty amino acids are converted

to the following seven compounds:

• Acetyl-CoA

•Acetatoacetyl-CoA

• Pyruvate

• Oxaloacetate

• -Ketoglutarate

• Succinyl-CoA

• Fumarate

Glucogenic Amino Acids Ketogenic Amino Acids

Metabolism of Carbon Skeletons of

Amino Acids

Citrate

OAA

Fumarate

Suc-CoA

-KG

Pyr Acetyl-CoA Acetoacetyl-CoA

Leu, Lys Phe, Tyr Trp

Glucose

Ile Leu Trp

Ala, Cys Gly, Ser Thr, Trp

Asp, Asn

Asp, Phe, Tyr

Ile, Met, Val

Arg Glu, Gln His, Pro

CO2

CO2

Entrance of Carbon Skeletons of Amino Acids into the Krebs Cycle

NH3+

C

H

COO-

H

NH3+

C

CH3

COO-

H

NH3+

CH COO-

CH2OH

NH3+

CH COO-

CH2SH

NH3+

C COO-

H

CHOH

CH3

H3C C COO-

O

CH2 C CH3

O

NH2

Glycine

Serine Alanine Cysteine Threonine

Pyruvate Aminoacetone

C3 Group

C4 Group

Asparagine

Keto Acid Amino Acid

NH4+

Aspartate Oxaloacetate

C5 Group

C H+H3N

COO-

CH2)2

COO-

( (

C

COO-

CH2)2

COO-

O

-KG Glutamate

Glutamine

Proline

Arginine

Histidine NH4

+

NH4+

C4 Group and C5 Group

Isoleucine Methionine Valine

Methylmalonyl-CoA

Succinyl-CoA

Propionyl-CoA

C S CoA

O

CH2CH2-OOC

C S CoA

O

C-OOC

CH3

H

CH3 CH2 C S CoA

OCarboxylase

Biotin

Mutase Vitamin B12

Propionyl-CoA is also generated from oxidation of odd-chained fatty acids

Deficiency of Vit B12 results in neurology problems in pernicious anaemia

Entrance Through Succinyl-CoA

Phenylalanine

CH2 CH

NH3

COO-

p-OH phenylpyruvate

Tyr transaminase -KG

CH2 C COO-

O

HO

Glu

Homogentisate

HO

OH

CH2 COO-

pHPP oxidase

CO2 O2

Fumarilacetoacetate

MAA isomerase

CH2 C

O

CH2 COO-CO

CH

CH

-OOC

Phe hydroxylase

Tyrosine

CH2 CH

HO

NH3

COO-

O2 + H4B

H2O + H2B

*

*

Maleilacetoacetate

Homogentisate oxidase

HC

HC

COO-

C

O

CH2 C

O

CH2 COO-

*

Fumarate

Acetoacetate

+

FAA hydrolase

C CCOO

-

-OOC H

H

H3C C

O

CH2 COO-

*

Entrance Through Fumarate

Phenylketonuria (PKU)

Hereditary disease that occurs1 in 10,000

Almost all untreated patients suffer mental retardation

Caused by deficiency of:

• Phe hydroxylase,

• Tetrahydrobiopterin, or

• Dihydrobiopterin reductase

Metabolites accumulated in patients’ plasma:

• Phenylalanine, Phenylpyruvate, Phenyllactate & Phenylacetate

Fair-coloured skin & hair (Phe/Tyr - precursor of melanin)

Diagnosis:

• Urine of neonate + FeCl3 → Phenylpyruvate (Green)

• Determination of plasma Phe levels

• DNA probe for Phe hydroxylase

Treatment: Diet low in Phenylalanine

Defects of the Phe Metabolism

Continuation…

Albinism

Lack of hair & skin pigment and photophobia mainly due to deficiency of tyrosinase, an enzyme involved in the conversion of Tyrosine to melanin.

Parkinson’s Disease

Progressive disease of the elderly, characterised by the tremor due to motor dysfunction at various muscles.

Caused by death of dopamine-producing cells in the substantia nigra & locus caeruleus.

Treatment involves administration of L-DOPA together with DOPA analogs that inhibit DOPA decarboxylase but are unable to cross the blood-brain barrier.

Defects of the Phe Metabolism

Valine Leucine Isoleucine

-ketoisocaproate

isovaleryl-CoA

Succinyl-CoA Acetyl-CoA + Acetoacetyl-CoA

Acetyl-CoA + Succinyl-CoA

Transaminase

-Keto acid dehydrogenase*

Acyl-CoA dehydrogenase

-ketoisovalerate -ketomethylvalerate

isobutyryl-CoA -methylbutyryl-CoA

CO2

CoA

FADH2

FAD

Glutamate

-Ketoglutarate

CH2 CHR:

CH3

CH3 CHR: CH3

CH3

CH3

CH3

CH2R: CH

*Maple syrup urine disease

Catabolism of Branched Chain

Amino Acids

A. Acids

The synthesis of glucose from non-carbohydrate metabolites.

Malate

Alanine

Malate OAA

OAA

(P)enolpyruvate

Pyruvate Carboxylase

Carboxykinase

Lactate

Asp/Asn

TCA Mitosol

Cytosol

3-(P)glycerate

2-(P)glycerate

1,3-bis(P)glycerate Glyceraldehyde -3(P)

Glucose

Glucose 6(P) Fructose 6(P)

Fructose 1,6bis(P)

Lactate

Glycerol + DHA(P)

Glc 6-phosphatase

Frc 1,6-bisphosphatase

Asp/Asn

ATP + CO2

ADP + Pi

GDP + CO2 GTP

Pi

Pi

Gluconeogenesis

Gluconeogenesis occurs mainly in the liver.

It is not a direct reversal of glycolysis.

Gluconeogenesis Glycolysis

Glucose 6-phosphatase Hexokinase

Fructose 1,6-bisphosphatase Phosphofruktokinase

Phosphoenolpyr Carboxykinase

Pyruvate Carboxylase Pyruvate Kinase

+ ATP + HCO3–

Pyruvate Carboxylase

Enzyme-Biotin

Pyruvate

+ ADP + Pi Enzyme-Biotin ~CO2

Enzyme-Biotin ~CO2 + Enzyme-Biotin +

Acetyl-CoA Mg2+

Mn2+

Oxaloacetate C

O

COO-

CH3-OOC CH2 C

O

COO-

Gluconeogenesis vs Glycolysis

Fructose 1,6-bisphosphatase is rate limiting.

Fate of oxaloacetate.

Malate OAA Pyr Carboxylase

ATP + CO2

ADP + Pi

Malate OAA

AMP

Frc 2,6-b(P)

Citrate

Fructose 1,6- bisphosphatase

NAD+ NADH

+ H+ NAD+ NADH + H+

TCA (P)enolpyruvate

Carboxy- kinase

Fructose 6-(P)

Fructose 1,6-bis(P)

Phospho- fructokinase

GO

GO

GO

Regulation of Gluconeogenesis

• Maintenance of blood glucose levels

• Relieves metabolic burden of muscle

Ala

Pyr

Glc

Trans- aminase Lac

Ala

Pyr

Glc

Pyr

Glc

Lac

(a) Glucose-alanine cycle (b) Cori’s cycle

LDH

Gluconeogenesis (6~P)

Glycolysis (2~P)

Glycolysis

-KG

Glu

NH4+

Urea

-KG

Glu

Protein

NAD+

NADH + H+ NAD+ NADH + H+

(2~P)

General Roles of Gluconeogenesis

Depending on their nutritional requirement, amino acids

may be classified as:

Essential

• Arg

• His

Non-essential

Semi-essential

• Ile

• Leu

• Lys

• Met

• Phe

• Thr

• Trp

• Val

• Ala

• Asp

• Asn

• Cys

• Gln

• Glu

• Gly

• Pro

• Ser

• Tyr

Requirement for Amino Acids

Glucose

Serine

Alanine

Cysteine

3-(P)glycerate

2-(P)glycerate

Pyruvate

H2SO4

Glycine COO

-

HC

CH2

OPO32-

OH

COO-

HC

CH2

OH

NH3+

COO-

HC

CH2

OPO32-

NH3+

Serine

3-(P)glycerate (P)OH-pyruvate

3-(P)serine

transaminase

phosphatase

deHase

COO-

HC

CH2

OPO32-

O

Amino Acids Derived from

Glycolysis

Continuation…

COO-

HC

CH2

OH

NH3+

Serine

S-adenosyl- methionine

S-adenosyl- homocysteine

Homocysteine

Methionine

Activated methyl cycle

~CH3

H2O -CH3

ATP

Homocysteine

H2O

Cystathionine

Cysteine -Ketobutyrate

HS CH2 CH COO-

NH3+

-OOC C CH2 CH3

O

-OOC CH CH2

NH3+

CH2 S CH2 CH COO-

NH3+

H2O

-OOC CH CH2

NH3+

CH2 SH

NH4+

+

+ Glycine

CO2 + NH4+ Serine

CH2THF + H2O

CH2THF THF

THF +H3N CH2 COO

-

Amino Acids Derived from

Glycolysis

Amino Acid Derived from Phe

Phenylalanine Phe hydroxylase

Tyrosine

O2 + H4B

Glucose

Proline

Aspartate

Glutamine

-KG

Pyruvate

Glutamate

Oxaloacetate

Aspartate Asparagine

Glu semialdehyde

Ornithine

Arginine

Urea

Asn Synthase

Gln Synthase

H2O + H2B

Amino Acids Derived from

Krebs Cycle

Creatine & Creatinine

C

NH

CH2

NHN2H

COOH

NH2

CH2

COOH

C

N

CH2

CH3

NHN2H

COOH

C

N

CH2

CH3

CHO

O

NHNHP

O

-O

O-

N

CN

C

C

NH

CH3

O

H

H H

Creatine

Creatinine

Guanidino acetate Glycine

Creatine phosphate

ATP

ADP

Arginine Ornithine SAM SAH

Pi CK

Biosynthesis of Specialised Products from Amino Acids

Continuation…

Glutathione (GSH)

g-Glutamyl- amino acid

Oxoproline

Amino Acid

(Cys, Gln)

Cysteinyl-glycine Dipeptidase

Oxoprolinase

g-Glutamylcyclo- transferase

g-Glutamyl- transpeptidase

Amino acid transport Reduction of MetHb → Hb Detoxification of H2O2 2GSH + ROOH → GSSG + H2O + ROH

Glutamate

g-Glutamyl-cysteine

Cysteine

g-Glutamyl-cysteine synthetase

ATP

ADP Glutathione Glycine

GSH synthetase ATP

ADP

Biosynthesis of Specialised

Products from Amino Acids

Thyroid Hormones

HO

CH2CH CO

NH

HO

CH2CH CO

NH

I

HO

CH2CH CO

NH

I

I

O

CH2CH CO

NH

I

I

I

O

CH2CH CO

NH

I

I

I

I

Tyrosine (TGB)

MonoiodoTyr

DiiodoTyr

Thyroxine

Triiodothyronine

H2O2 + H+ + I–

2H2O

H2O2 + H+ + I–

2H2O

DiiodoTyr

Alanine

IODINASE

Continuation…

Neurotransmitters, Hormones & Skin Pigment

HO CH2 C

NH3+

H

COO-

HO

HO CH2

HO

CH2NH3+

HO CH

HOOH

CH2NH3+

HO CH

HOOH

CH2NHCH3

HO CH2 C

NH3+

H

COO-

Tyrosine

DOPA Dopamine

Norepinephrine Epinephrine

Tyr hydroxylase

Tyrosinase

Decarboxylase

Oxidase

Methyl transferase

Eumelanins, Pheomelanins &

Mixed-typed Melanins O2 + H4B

H2O + H2B

SAM SAH

CO2

O2

Continuation…

Thank you