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