Amino Acid Metabolism

Hanley N. Abramson
Professor of Pharmaceutical Sciences
Wayne State University
December 2009
 Dynamics of Protein
And Amino Acid Metabolism

Dietary Proteins Digestion to Amino Acids

Transport in Blood to Cells

Protein Synthesis Functional Proteins

Amino Acids Protein Degradation In

Proteasomes Following
Tagging With Ubiquitin
Metabolites 2
 Digestion of Proteins
Stomach: Pepsinogen Pepsin (max. act. pH 2)
Enteropeptidase
Small Intestine: Trypsinogen Trypsin

Trypsin cleaves:
Chymotrypsinogen to chymotrypsin
Proelastase to elastase
Procarboxypeptidase to carboxypeptidase

Aminopeptidases (from intestinal epithelia)

3
 Intestinal Absorption
Amino Acids Oligopeptides
Lumen

Transport
Protein
Oligopeptides

Peptidases

Amino Acids

Blood
4
 +
Incorporation of NH4 Into
Organic Compounds
Carbamoyl
+ -
1) NH4 + HCO3 + 2 ATP Phosphate NH2CO2PO3-2 + 2 ADP +
Synthase I
+
(CPS-I) Carbamoyl Phosphate Pi + 2 H

TCA Cycle mitochondria

O Glutamate NH3 +
-
dehydrogenase
+ O 2 CCH 2 CH 2 CCO 2 - -
O 2 CCH 2 CH 2 CHCO 2 -
2) NH4 +
-Ketoglutarate NADPH + NADP+ Glutamate
H+

5
 +
Incorporation of NH Into 4
Organic Compounds (Cont.)

3) NH3 +
-
O 2 CCH 2 CH 2 CHCO 2 - + NH4+ + 2 ATP
Glutamate Glutamine
Synthase
Mg++

O NH3+
H2 NCCH 2 CH 2 CHCO 2 -

Glutamine
N of glutamine donated to other compounds
in synthesis of purines, pyrimidines,
6
and other amino acids
 Biosynthesis of Amino Acids:
Transaminations
Amino Acid1 +-Keto Acid2 Amino Acid2 +-Keto Acid1

NH3 + O
-
O 2 CCH 2 CH 2 CHCO 2 - + R-CCO 2 -
Glutamate Pyridoxal phosphate (PLP)-
Dependent Aminotransferase

O
NH2
-
O 2 CCH 2 CH 2 CCO 2 - +
R-CHCO 2 -
-Ketoglutarate
7
Transaminations: Role of PLP
CO2-

CHO H N CHCH2CH2CO2-
C
HO CH2OPO3-2
HO CH2OPO3-2

H3 C
N H3 C
+ N
H +
NH3 + H2 O H
-
O 2 CCH 2 CH 2 CHCO 2 - Tautomerization

O CO2-
-
O 2 CCH 2 CH 2 CCO 2 - N CCH2 CH2CO2-
CH2NH2 CH2
HO CH2OPO3-2 HO CH2 OPO3-2

H3 C N H2 O H3C
+ N
+
H H 8
 Transaminations
Glutamate-Pyruvate
Aminotransferase
Glutamate (Alanine Transferase ALT) -Ketoglutarate
+ +
Pyruvate Alanine
Glutamate-Oxaloacetate
Aminotransferase
(Aspartate Transferase AST)
Glutamate -Ketoglutarate
+ +
Oxaloacetate Aspartate

Blood levels of these aminotransferases, also called transaminases,

are important indicators of liver disease

9
Metabolic Classification of
the Amino Acids
• Essential and Non-essential

• Glucogenic and Ketogenic

10
 Non-Essential Amino Acids
in Humans
• Not required in diet
• Can be formed from -keto acids by
transamination and subsequent
reactions • Alanine • Glycine
• Asparagine • Proline
• Aspartate • Serine
• Glutamate • Cysteine (from Met*)
• Glutamine • Tyrosine (from Phe*)
* Essential amino acids
11
 Essential Amino Acids in
Humans
• Required in diet
• Humans incapable of forming requisite
carbon skeleton
• Arginine* • Lysine
• Histidine* • Methionine
• Isoleucine • Threonine
• Leucine • Phenylalanine
• Valine • Tryptophan

* Essential in children, not in adults

12
 Glucogenic Amino Acids
• Metabolized to -ketoglutarate, pyruvate,
oxaloacetate, fumarate, or succinyl CoA
Phosphoenolpyruvate Glucose

• Aspartate • Methionine • Alanine

• Asparagine • Valine • Serine
• Arginine • Glutamine • Cysteine
• Phenylalanine • Glutamate • Glycine
• Tyrosine • Proline • Threonine
• Isoleucine • Histidine • Tryptophan

13
 Ketogenic Amino Acids
• Metabolized to acetyl CoA or acetoacetyl
CoA

Animals cannot convert acetyl CoA or

acetoacetyl CoA to pyruvate
• Isoleucine • Tryptophan
• Leucine * • Phenylalanine
• Lysine * • Tyrosine
• Threonine
* Leucine and lysine are only ketogenic
14
 Amino Acids Formed From
-Ketoglutarate
O
-
O 2 CCH 2 CH 2 CCO 2 -
-Keto- 4 Steps
Transamination or
glutarate Glutamate
dehydrogenase
+ CO2-
N
NH3 +
H H Proline
- - 5 Steps
O 2 CCH 2 CH 2 CHCO 2
Glutamate NH3 +
+
Glutamine
H3 NCH 2 CH 2 CH 2 CHCO 2 - Ornithine
synthase
Urea Cycle
O NH3+ NH2 NH3 +
+
H2 NCCH 2 CH 2 CHCO 2 - H2 N=C-HNCH 2 CH 2 CH 2 CHCO 2 - Arginine
Glutamine 15
Guanidino group
 GABA Formation
Glutamate
NH3 + NH3 +
decarboxylase
-
O 2 CCH 2 CH 2 CHCO 2 - -
O 2 CCH 2 CH 2 CH 2
Glutamate Gamma-aminobutyrate
CO2 (GABA)

GABA is an important inhibitory neurotransmitter

in the brain
Drugs (e.g., benzodiazepines) that enhance the effects
of GABA are useful in treating epilepsy
16
 Arginine Synthesis: The Urea Cycle

NH3 + N-Acetylglutamate NHCOCH3

- - synthase
O 2 CCH 2 CH 2 CHCO 2
-
O 2 CCH 2 CH 2 CHCO 2
-
CoASAc
Glutamate N-Acetylglutamate
Activates
4 Steps

Carbamoyl NH3 +
CPS-I phosphate +
NH4+ + HCO3- NH2CO2PO3-2 H3 NCH 2 CH 2 CH 2 CHCO 2 -
Ornithine

NH3+ Ornithine
Transcarbamoylase (OTC)
NH2 CONH CH 2 CH 2 CH 2 CHCO 2 - (mitochondria)
Citrulline
Ureido group 17
 The Urea Cycle (Contd.)
NH3 + + Asp NH3 +
NH3 -CHCH2CO2 - +
-
NH2 CONH CH 2 CH 2 CH 2 CHCO 2 CO2 - H2 N=C-HNCH 2 CH 2 CH 2 CHCO 2 -
Citrulline
Arginosuccinate NH-CHCH2 CO2-
synthase
CO2 - Arginosuccinate
Ornithine
Transcarbamoylase
(mitochondria) H CO2 -

Fumarate
NH3 + Urea
+ -O
2C H

H3 NCH 2 CH 2 CH 2 CHCO 2 - H2NCONH2 Argino-

succinase
Ornithine TCA Cycle
NH2 NH3 +
Arginase +
These reactions occur H2 N=C-HNCH 2 CH 2 CH 2 CHCO 2 -
in the cytosol 18
Arginine
 Urea Formation
• Occurs primarily in liver; excreted by kidney
• Principal method for removing ammonia
• Hyperammonemia:
• Defects in urea cycle enzymes (CPS, OTC, etc.)
• Severe neurological defects in neonates
• Treatment:
» Stop protein intake
» Dialysis
» Increase ammonia excretion: Na benzoate, Na
phenylbutyrate, L-arginine, L-citrulline

19
 Blood Urea Nitrogen
• Normal range: 7-18 mg./dL
• Elevated in amino acid catabolism
• Glutamate N-acetylglutamate
CPS-1 activation
• Elevated in renal insufficiency
• Decreased in hepatic failure

20
Synthesis of Nitric Oxide
NH2 NH3 +
+ Arginine
H2 N=C-HNCH 2 CH 2 CH 2 CHCO 2 -

Nitric oxide synthase (NOS)

NH3 +
NH2 CONH CH 2 CH 2 CH 2 CHCO 2 - + NO

Citrulline

21
 Nitric Oxide
• Cell messenger
• Implicated in a wide range of physiological
and pathophysiological events:
• Vasodilation:
• Activates guanylyl cyclase cGMP
• Nitroglycerin Glycerin + NO
• Sildenafil (Viagra): in vascular smooth muscle:
Blocks Phospho-
diesterase-5
NO cGMP GMP

22
 Formation of Serine
CO 2 - CO 2 -
Dehydrogenase
Glycolysis
Glucose H C OH C=O
NAD+ NADH +
CH2 OPO3-2 H+
CH2OPO3-2
3 Steps 3-Phospho- 3-Phospho-
glycerate hydroxypyruvate
Pyruvate
Inhibits Glutamate
Transaminase
-Ketoglutarate
-
CO 2 CO 2 -
Phosphatase
H C NH3 +
H C NH3 +

CH2 OH CH2 OPO3-2

Serine (Ser) 3-Phosphoserine
23
Conversion of Serine to Glycine
H

Dihydrofolate H2N N N
reductase
CO 2 -
Folate N
N CH2 NHR
H C NH3+
OH
H Serine
Tetrahydrofolate CH2 OH
(FH4)
Serine hydroxymethyl
transferase (PLP-dep.)
H
N
Key intermediate CO 2 -
in biosynthesis of N CH2 Glycine
H C NH3 +
purines and N
formation of H2 C H
thymine Important in
N5, N10-Methylene FH4
biosynthesis of heme,
porphyrins, and purines
24
 Sarcosine (N-Methylglycine)
O
O Glycine N-Methyltransferase
H2
N
H3N H3C O
O
Glycine Sarcosine

Sarcosine as a possible biomarker for prostate cancer:

See Nature Feb. 12, 2009

25
 Sulfur-Containing Amino Acids
Methionine
Synthase NH3+
NH3 + (Vit. B12-dep.) -
- HSCH 2CH 2CHCO 2 + 5-Methyl
CH 3SCH 2CH 2CHCO 2 + FH4 FH4
Methionine L-Homocysteine
(Essential) CO 2 -
Cystathionine
H C NH3 + Serine
-synthase
CH2 OH
(PLP-dep.)

Cystathionine NH3 +
OH NH3 + lyase -
SCH 2CH 2CHCO 2
- -
CH 3CHCH 2CO 2 + HSCH 2CHCO 2 CH2 CHCO2 -
Cystathionine
-Hydroxy- Cysteine NH3+
butyrate (Non-essential)
26
 Homocysteine
Homocysteinuria
• Rare; deficiency of cystathionine -synthase
• Dislocated optical lenses
• Mental retardation
• Osteoporosis
• Cardiovascular disease death

High blood levels of homocysteine associated with

cardiovascular disease
• May be related to dietary folate deficiency
• Folate enhances conversion of
homocysteine to methionine
27
 Methionine Metabolism:
Methyl Donation NH2
N
N

S-Adenosyl methionine N N
NH3 +
synthase +
- -O CCHCH CH -S-H2C
CH 3SCH 2CH 2CHCO 2 2 2 2 O

ATP NH3 + CH3

Methionine
OH OH
SAM
Decarboxylase S-Adenosyl
Methionine
NH2
NH2 (SAM)
N
N N
Decarboxylated N
CO2
SAM N
R-H
N
N N Methyl-
-O transferases
+ + 2 CCHCH2 CH2 -S-H2 C O
H3NCH2CH2CH 2-S-H2C O NH3 + R-CH3
CH3
S-Adenosyl
OH OH
homocysteine 28
OH OH
 Polyamine Biosynthesis
Ornithine
decarboxylase
NH3 + +
+ (ODC)
+ NH3
H3 NCH 2 CH 2 CH 2 CHCO 2 - (PLP-dep.)
H3 N
Ornithine Putrescine
(from urea cycle) CO2 Decarboxylated
SAM
Spermidine
H
+ synthase
+ + N+ NH3
H3 N N 5’-Methylthio-
H Spermine
H H adenosine
synthase
Spermine
5’-Methylthio- H +
+ +N NH3
adenosine H3 N H
Decarboxylated Spermidine
SAM
29
 Polyamines
• Spermidine and spermine found in virtually
all procaryotic and eucaryotic cells

• Precise role undefined

• Bind to nucleic acids

• Inhibition of biosynthetic pathway:

CO2 H
-Difluoromethyl-
NH2
ornithine (DFMO)
H2 N
(Eflornithine) - inhibits ODC;
CHF2 used to treat
Pneumocystis carinii infectons
30
 Creatine and Creatinine
Arginine-glycine
transamidinase NH2
NH2 NH3 + (Kidney) +
+
H2 N=C-HNCH 2 CH 2 CH 2 CHCO 2 - H2 N=C-HNCH 2 CO 2 -
Glycine Ornithine
Arginine Guanidoacetate
SAM + ATP
H Guanidoacetate
N O Methyltransferase S-Adenosyl-
(Liver) homocysteine
Creatinine HN + ADP

(Urine) N Non-enzymatic
(Muscle) NHPO3 -2
CH3 +
Creatine kinase
H2 N=C-NCH 2 CO 2 -
NH2 (Muscle) CH3
+
Creatine H2 N=C-NCH 2 CO 2 - ADP
Phosphocreatine
+ Pi
CH3 ATP
31
 Creatine and Creatinine
Creatine:
• Dietary supplement
• Used to improve athletic performance

Creatinine:
• Urinary excretion generally constant;
proportional to muscle mass

Creatinine Clearance Test:

• Compares the level of creatinine in urine (24 hrs.)
with the creatinine level in the blood
• Used to assess kidney function
• Important determinant in dosing of several drugs
in patients with impaired renal function

32
 Histidine Metabolism:
Histamine Formation
+
NH3
H
N CH2 CHCO2 - Histidine H
decarboxylase N CH2 CH2NH2

N
N
Histidine CO2 Histamine
Histamine:
• Synthesized in and released by mast cells
• Mediator of allergic response: vasodilation, bronchoconstriction
(H1 receptors)
• H1 blockers: Diphenhydramine (Benadryl)
Loratidine (Claritin)
• Stimulates secretion of gastric acid (H2 receptors)
• H2 blockers: Cimetidine (Tagamet); ranitidine (Zantac)
33
 Phenylalanine and Tyrosine
Phenylalanine H
H2N N N H
(Essential)
Tetrahydrobiopterin
H
NH3+ + HN
(BH4)

N CHCHCH 3
CH2 CHCO2-
O H HO OH
O2 Phenylalanine-4- NADP+
Monooxygenase
(Phenylalanine
H2O NADPH + H+
hydroxylase)
HO N N
H2N
NH3 + Dihydrobiopterin
+
CH2CHCO2- HN
N CHCHCH 3
Tyrosine H
(Non-essential)
O HO OH

34
Normal Utilization of Phenylalanine

Protein (~25%)
Phenylalanine
Tyrosine (~75%)

35
Phenylketonuria (PKU) Disease
• Deficiency of Phe hydroxylase
• Occurs in 1:20,000 live births in U.S.
• Seizures, mental retardation, brain
damage
• Treatment: limit phenylalanine intake
• Screening of all newborns mandated
in all states Tyr

Phe Phenylpyruvate
Transamination (urine)
O
36
CH2CCO2-
 Sapropterin (Kuvan)

• Synthetic tetrahydrobiopterin
• Indicated for reduction of phenylalanine blood
levels in patients with BH4-responsive PKU

37
 Catecholamine Biosynthesis
HO Catechol
HO
Tyr hydroxylase
NH3+ NH3+
CH2 CHCO2- O2 HO CH2 CHCO2-

Tyrosine Dihydroxyphenylalanine
(DOPA)
HO DOPA
Epinephrine
(Adrenaline) decarboxylase CO2
HO CHCH2 NHCH3
HO
OH Methyl Dopamine
transferase hydroxylase
S-Adenosyl- HO HO CH2 CH2 NH2
homocysteine
SAM Dopamine

HO CHCH2 NH2
DOPA, dopamine, norepinephrine, OH
Norepinephrine 38
and epinephrine are all neurotransmitters
 L-DOPA in Parkinsonism
Blood Brain

L-DOPA L-DOPA Dopamine

HO
CH3
Blocks
HO
CH2-C-CO2 H

Carbidopa
NHNH2 Parkinsonism associated with
dopamine in brain through loss of
Dopamine neurons in basal ganglia.
Carbidopa + L-DOPA

Blood Brain Barrier 39

 Monoamine Oxidase (MAO)
HO MAO HO
(in mitochondria)
HO CHCH2NHR' CHCHO
HO
R R

R R’ Aldehyde
dehydrogenase
OH H Norepi
HO
OH CH3 Epi
H H Dopamine HO CHCO2 H

Urinary R
MAO inhibitors (e.g., tranylcypromine) are useful
metabolite
in the treatment of depression
R=OH Vanillylmandelic acid (VMA)
Brain levels of dopamine and norepi.; also R=H Homovanillic acid (HVA)
serotonin
40
 Tyramine
OH OH
MAO

CH2 CH2 NH2 CH2CHO

Tyramine
( blood pressure)

• Tyramine found naturally in several types of cheese;

also beer and red wine.
• Tyramine intake can cause hypertensive crisis in
persons taking a MAO inhibitor ( norepi release)
41
 Catechol-O-Methyl
Transferase (COMT)
HO HO
COMT
HO CHCH2NHR' CHCH2NHR'
CH3 O
SAM S-Adenosyl-
Active R homocysteine R
catecholamine Inactive
metabolite
• COMT found in cytoplasm
• Terminates activity of catecholamines
• Catecholamine excretion products result from
combined actions of MAO and COMT
• Inhibitors of COMT (e.g., tolcapone) useful
in Parkinson’s disease 42
Homogentisic Acid Formation
HO HO
Transamination O
NH3+
CH2 CHCO2- CH2CCO2-

Tyrosine p-Hydroxyphenyl-
pyruvate
Deficient in O2
alkaptonuria p-Hydroxyphenyl-
pyruvate
OH
Homogentisate dioxygenase
dioxygenase (ascorbate-dep.)
Cleavage of
aromatic ring CO2
CH2CO2-
O2
Fumarate + acetoacetate OH
Homogentisate 43
 Alkaptonuria
• Deficiency of homogentisate dioxygenase

• Urine turns dark on standing

• Oxidation of homogentisic acid

• Asymptomatic in childhood

• Tendency toward arthritis in adulthood

44
 Melanin Formation
HO
HO
Tyr hydroxylase
NH3+
NH3+
CH2 CHCO2 - O2 HO CH2 CHCO2-
Tyrosine DOPA
Tyrosinase

O
Melanin Highly colored
(Black polymer) polymeric
intermediates O CH2 CHCO2 -
NH3
+
Melanin formed in skin (melanocytes), eyes, and hair
In skin, protects against sunlight Dopaquinone
Albinism: genetic deficiency of tyrosinase
45
 Tryptophan Metabolism:
Serotonin Formation
+
Indole ring NH3 +
NH3
CH2 CHCO2 -
CH2 CHCO2 - CH2 CH2 NH2
Trp
hydroxylase HO HO
Decarboxylase
N
H O2 N
N
H H
Tryptophan 5-Hydroxy- CO2 5-Hydroxy-
(Trp) tryptophan tryptamine (5-HT);
Serotonin

46
 Serotonin
• Serotonin formed in:
• Brain (neurotransmitter; regulation of sleep, mood, appetite)
• Platelets (platelet aggregation, vasoconstriction)
• Smooth muscle (contraction)
• Gastrointestinal tract (enterochromaffin cells - major storage site)

• Drugs affecting serotonin actions used to treat:

• Depression
•Serotonin-selective reuptake inhibitors (SSRI)
• Migraine
• Schizophrenia
• Obsessive-compulsive disorders
• Chemotherapy-induced emesis

• Some hallucinogens (e.g., LSD) act as serotonin agonists

47
 L-Tryptophan
• Food supplement promoted for serotonin effects
• L-Tryptophan disaster (1989):
• Eosinophilia-myalgia syndrome (EMS)
• Severe muscle and joint pain
• Weakness
• Swelling of the arms and legs
• Fever
• Skin rash
• Eosinophilia
• Many hundreds of cases; several deaths
• Traced to impurities

48
 Serotonin Metabolism: 5-HIAA
CH2 CH2NH2 CH2CHO
HO MAO HO

N N
H H

Serotonin Dehydrogenase
CH2 CO2H
HO

Carcinoid tumors:
• Malignant GI tumor type N
H
• Excretion of large amounts of 5-HIAA
5-Hydroxyindole acetic
acid (5-HIAA) (Urine) 49
 Serotonin Metabolism:
Melatonin
CH2 CH2 NH2 CH2CH2 NHCOCH3
HO H3CO
2 Steps
N N
H H
Serotonin Melatonin
Melatonin:
• Formed principally in pineal gland
• Synthesis controlled by light, among other factors
• Induces skin lightening
• Suppresses ovarian function
• Possible use in sleep disorders
50
 Tryptophan Metabolism:
Biosynthesis of Nicotinic Acid
+
NH3

CH2 CHCO2 - CO2H

Several steps

N
N
H
Tryptophan Nicotinic acid (Niacin)

Nicotinamide adenine
dinucleotide (NAD)
51