1

AMINO ACIDS 2
GOALS/OBJECTIVES
At the end of this lecture students will be able
to:
Differentiate between protein and non-protein
amino acids;
Describe the functions of the different non-
protein amino acids;
Describe the processes of transamination and
deamination;
Describe the processes of the urea cycle and
urea synthesis;

2
Identify amino acid anabolic derivatives
and their functions;
Describe and differentiate amino acid-related
diseases.
3
NON-PROTEIN AMINO ACIDS.

The clinically significant non-protein amino acids
include the following:

citrulline, ornithine, carnitine, creatine and
taurine.

However, this list is not exhaustive and others do
have some clinical significance, but usually in
highly specialized areas or in minor pathways.


.
4
CITRULLINE

Free citrulline is mainly made from ornithine and
carbamoyl phosphate in one of the central
reactions in the urea cycle. Citrulline in proteins
has been produced from arginine.
Patients with rheumatoid arthritis
often have detectable antibodies
against proteins containing citrulline.
Detection of antibodies reactive with
citrulline-containing proteins or
peptides is now becoming an important
help in the diagnosis of rheumatoid
arthritis. In recent studies,
citrulline has been found to
relax blood vessels.

.
5
ORNITHINE

Ornithine is a central part of the urea cycle,
which allows for the disposal of excess nitrogen.
First, ammonia is converted into carbamoyl
phosphate onto which ornithine is added. Another
nitrogen is added from aspartate, producing
fumarate, and then arginine, which is hydrolysed
back to ornithine, producing urea. In mammalian
non-hepatic tissues, the main use of the urea
cycle is in arginine biosynthesis, so as an
intermediate in
metabolic processes,
ornithine is quite
important.
6
CARNITINE

Carnitine is synthesized primarily in the liver and
kidneys from the amino acids lysine or methionine.
Vitamin C (ascorbic acid) is essential to the
synthesis of carnitine. Carnitine transports long-
chain fatty acids into the mitochondrial matrix, so
that they can be broken down through -oxidation
to acetylCoA to obtain usable energy via the TCA
cycle. Carnitine also has antioxidant properties,
providing protection against lipid peroxidation of
membranes.
7
CREATINE

Creatine occurs in vertebrates and about half of
the creatine originates from food, while the rest
is synthesized from arginine, glycine, and
methionine. It functions as creatine phosphate to
provide short term energy. Creatinine is a break-
down product of creatine phosphate and is usually
produced at a fairly constant rate
by the body. Chemically, creatinine
is a cyclic derivative of creatine.
Creatinine is filtered by the kidneys
into the urine. There is no tubular
reabsorption of creatinine. So,
creatinine levels in blood and urine
may be used to calculate the
glomerular filtration rate (GFR).
8
TAURINE

Taurine is a derivative of cysteine, and is
conjugated with chenodeoxycholic acid and cholic
acid to form the bile salts. Taurine crosses the
blood-brain barrier and is an inhibitory
neurotransmitter. It also acts as an antioxidant
and protects against toxicity of various heavy
metals. Taurine is thought to be a dietary
essential nutrient in infants and has been added
to many infant formulas.
9
TRANSAMINATION AND DEGRADATION




AMINO ACIDS



GLUTAMATE



UREA
10
Excess amino acids need to be removed from the
body, but are not usually excreted in urine.
Detection of amino acids in urine usually reflects
either kidney or metabolic pathology. The prime
mechanism for amino acid removal is for the
amine group to be moved to another carbon
backbone. This backbone is most often -
ketoglutarate and the aminated product is
glutamate.

This moving of the amine is termed
transamination or aminotransfer and is usually the
first step in their degradation. All the amino
acids have specific transaminase enzymes which
convert them to glutamate and their de-aminated
-keto acid carbon backbone.
11






H
|
HOOC C NH
2
HOOC C = O
| |
X X
12
All the transaminase enzymes require pyridoxal
phosphate (vitamin B6) as coenzyme and during
the reaction it accepts the amine from the amino
acid, forming the -keto acid and becoming
pyridoxamine, then the amine is donated to -
ketoglutarate forming glutamate and pyridoxal
phosphate again.

Since quantitatively alanine and aspartate are two
of the most common amino acids, as well as being
closely linked to energy metabolism, the
transaminase enzymes that convert them to
glutamate are the most common, especially in
liver.


13
GENERICALLY:

AMINO ACID + -KETOGLUTARATE (KG) -KETO ACID (KA) + GLUTAMATE

(PYRIDOXAL PHOSPHATE PYRIDOXAMINE PYRIDOXAL PHOSPHATE)

TRANSAMINASE TRANSAMINASE


SO:

'X' (WITH NH
2
) + KG (WITHOUT NH
2
)
'Y' (WITHOUT NH
2
) + GLUTAMATE (WITH NH
2
)

14

SPECIFICALLY IN LIVER:

ALANINE + -KETOGLUTARATE PYRUVATE + GLUTAMATE
ALANINE TRANSAMINASE









COOH COOH
l l
CH
2
CH
2
l l
CH
3
CH
2
CH
3
CH
2
l l l l
CH-NH
2
+ C=O C=O + CH-NH
2
l l l l
COOH COOH COOH COOH
15
ASPARTATE + -KETOGLUTARATE OXALOACETATE + GLUTAMATE
ASPARTATE TRANSAMINASE


COOH COOH
l l
COOH CH
2
COOH CH
2
l l l l
CH
3
CH
2
CH
3
CH
2
l l l l
CH-NH
2
+ C=O C=O + CH-NH
2
l l l l
COOH COOH COOH COOH
16
This means that all amino acids can be converted to
one common intermediate amino acid, which can then
be metabolised and feed the amine into the urea
cycle for urea synthesis. The -keto acid carbon
backbones feed into the energy generation/fuel
synthesis pathways at some point or other, depending
on their structure.

The problem with this process is that -ketoglutarate
levels in the cell are limited, so to keep the process
going requires it to be regenerated. This is
accomplished by linking the transaminase reactions to
glutamate dehydrogenase, which converts glutamate
back to -ketoglutarate and releases NH
4
+
for the
urea cycle.


17
Quantitatively, most of this process occurs in
the liver, and the NH
4
+
is fed into the urea
cycle for conversion to urea and ultimate
excretion through the kidneys.


COOH COOH
l l
CH-NH
2
C=O
l l
CH
2
+ H
2
0 CH
2
+ NH
3
/NH
4
+

l l
CH
2
CH
2
l l
COOH COOH
GLUTAMATE + WATER -KETOGLUTARATE + AMMONIA
18

PUTTING IT ALL TOGETHER:

AMINO ACID + -KETOGLUTARATE GLUTAMATE + -KETO ACID

(PYRIDOXAL PHOSPHATE PYRIDOXAMINE PYRIDOXAL PHOSPHATE)

TRANSAMINASE TRANSAMINASE



-KETOGLUTARATE + NH
4
+
+ NADH
2
GLUTAMATE + NAD +H
2
0
GLUTAMATE DEHYDROGENASE
UREA


19
amino acid urea
+ +
KG KG


glutamate glutamate
+
KA

GDH
ALT/AST
Or another way of showing the same thing:
20
A further effect of transamination is that the non-
essential amino acids can to varying degrees be
interconverted, as the carbon backbone can be
synthesised and then an amine added via glutamate,
and the appropriate transaminase, from another
amino acid. This does not apply to the essential
amino acids as humans cannot synthesise their
carbon backbones.

Amino acids are classified as being either glucogenic
or ketogenic. Glucogenic amino acids can be
converted to an intermediate in glucose metabolism.
Ketogenic amino acids can be converted to
acetylCoA or acetoacetylCoA, but not glucose.
Mixed amino acids can be converted to both a
glucose intermediate as well as acetoacetylCoA or
acetylCoA.
21

Glucogenic amino acids are: aspartate,
asparagine, glutamate, glutamine, histidine,
proline, arginine, glycine, alanine, serine,
cysteine, methionine and valine.

Ketogenic amino acids are: leucine and lysine.

Mixed amino acids are: phenylalanine, tyrosine,
tryptophan, isoleucine and threonine.
22
GLUCOSE GLY, CYS, SER, THR, TRY ISO, LEU, LEU, LYS, PHE,
TRY TYR, TRY
ALA
PEP
PYRUVATE ACETYLCoA ACETOACETYLCoA


ASP, OXALOACETATE
ASN LIPIDS
TCA CYCLE
TYR, FUMARATE CITRATE ACETYLCoA
PHE,
ASP

ISO, -KETOGLUTARATE GLU, GLN,
MET, SUCCINYLCoA
VAL HIS, PRO, ARG
23
UREA CYCLE

Urea is produced in the liver by a very efficient
process that minimises the amount of carbon
lost to transport the ammonia. As with most
pathways the first step is the rate-limiting
step, and produces carbamoyl phosphate from
glutamate-derived ammonia and bicarbonate.
The enzyme is carbamoyl phosphate synthetase,
the reaction costs 2 ATP, and it occurs inside
the mitochondria. Carbamoyl phosphate is then
condensed with ornithine via ornithine
transcarbamoylase and this produces citrulline,
which then diffuses out of the mitochondria.
In the cytosol, argininosuccinate synthetase
combines citrulline with aspartate to form
arginosuccinate. This also costs 1 ATP.
24
Both the amine groups of urea are now in place,
one from ammonium and one from aspartate.
Arginosuccinase now cleaves the arginosuccinate
to form arginine and fumarate. The fumarate
recycles back to aspartate via oxaloacetate and
can be reused in the cytosol. The arginine is
then split via arginase to form urea and
ornithine. The urea diffuses out of the liver cell
and is transported in solution to the kidneys for
excretion, the ornithine is retro-transported
back into the mitochondria where the cycle can
carry on again.

25
NH
4
+
HCO
3
-
MITOCHONDRION
CARBAMOYL PHOSPHATE

ORNITHINE


CITRULLINE

ASPARTATE

CYTOSOL ARGININOSUCCINATE

OXALOACETATE
ARGININE
FUMARATE

UREA
PLASMA
26
NH
2


C=O

NH
2


UREA

So the liver has taken a mixed pool of
excess amino acids and converted them all to
energy metabolism intermediates and a single
excretory product, urea.
27
The primary regulatory mechanism for the urea
cycle is the concentration of the substrate for
carbamoyl phosphate synthetase, the ammonia
derived via glutamate deamination. The enzymes
of the urea cycle are also induced or suppressed,
depending on whether the diet is high or low in
protein.
Given the central role of the urea cycle in
metabolism, defects in any of the enzymes are
often fatal. Development in utero is usually
normal, as the placenta and mother's circulation
remove the excess ammonia. Post natal
symptomology often develops rapidly and
commonly causes early death.
28
CORI, GLUCOSE-ALANINE CYCLE
Under aerobic conditions, ie. when the muscle is
working, glycogenolysis and glycolysis provide
substrates for the TCA cycle and ETC to make ATP
for the muscle itself.
When the muscle is resting, its energy requirements
are very low, so the energy released from just
glycolysis, ie. anaerobic, is adequate to fuel its
requirements.
However, this produces a lot of lactate which needs
to be removed. It diffuses out of the cell into the
circulation and goes to the liver. In the liver it is
converted back to pyruvate, and this goes via
gluconeogenesis to glucose. This is the Cori cycle.

29
GLUCOSE
GLUCOSE
LACTATE
LACTATE
LACTATE
PYRUVATE
LDH
LDH
FATTY
ACIDS
GLYCEROL
-OXIDATION
LIVER
MUSCLE
ADIPOSE
RBCS
CORI CYCLE
PYRUVATE
30
Another cycle is going on at the same time. Under
anaerobic conditions, protein turnover still
continues, producing ammonia. This is converted to
glutamate, which is then converted to alanine via
transamination of some of the pyruvate produced
from glycolysis. This alanine is then exported and
sent to the liver, where it undergoes reverse
transamination to produce pyruvate and glutamate
again. The pyruvate then feeds into
gluconeogenesis to produce glucose again, which
recycles back to the muscle, while the glutamate
feeds its ammonia into the urea cycle. Thus there
is a continual recycling of 'waste products' from
muscle to be recycled in the liver. This is the
glucose-alanine cycle.
31
GLUCOSE
GLUCOSE
ALANINE
ALANINE
ALANINE
PYRUVATE
PYRUVATE
ALT
ALT
LIVER
MUSCLE
GLUCOSE-ALANINE CYCLE
32
GLUCOSE
GLUCOSE
LACTATE
LACTATE
LACTATE
PYRUVATE
PYRUVATE
LDH
LDH
FATTY
ACIDS
GLYCEROL
-OXIDATION
LIVER
MUSCLE
ADIPOSE
RBCS
COMBINED CYCLES
ALANINE
ALANINE
ALT
ALT
33
HEME SYNTHESIS
The synthesis of the heme group of hemoglobin,
myoglobin and the cytochromes involves glycine
directly, and several other amino acids indirectly.
The process requires glycine to be condensed onto
succinylCoA to form 5()-aminolevulinic acid
(ALA). 2 of these form a dimer and then a
pyrrole ring, porphobilinogen. 4 of these rings
combine together to ultimately produce heme.
SuccinylCoA can be produced from isoleucine,
methionine and valine directly, or indirectly from
glutamine, glutamate, histidine, proline and
arginine via -ketoglutarate. SuccinylCoA is also a
key intermediate in the TCA cycle, thus heme
synthesis is closely linked to energy metabolism.
34
SUCCINYLCoA
SUCCINATE

GLYCINE
PORPHOBILINOGEN
PROTOPORPHYRIN
HEMOGLOBIN
Fe
2+
35
AMINO ACID DERIVATIVES.

There are a range of derivatives of amino acids
other than proteins and degradation products.

These fall into 2 categories:

Hormone derivatives, and
neurotransmitter derivatives.

Hormone derivatives are made from tyrosine.

Neurotransmitter derivatives are, or are made
from, glycine, aspartate, glutamate, choline,
histidine, tyrosine and tryptophan.
36
HORMONE DERIVATIVES.

Thyroid hormones (t3 and t4).
Thyroid hormone synthesis starts with
thyroglobulin. This is a large glycoprotein with 2
joined units. Iodine is incorporated into specific
tyrosine residues at certain sites within the
thyroglobulin structure. The iodinated
thyroglobulin is then stored. When needed the
thyroglobulin is degraded by lysosomal
proteases. This produces either mono-
iodotyrosine (MIT) or di-iodotyrosine (DIT).
Lastly, the iodinated tyrosines are brought
together to form the t3 or t4.
1 MIT + 1 DIT = t3, 1 DIT + 1 DIT = t4.
37
DIT DIT MIT DIT
DIT MIT MIT MIT
38
ADRENALINE.

Tyrosine is converted to dihydroxy-phenylalanine
(DOPA) by tyrosine hydroxylase. This is then
converted to dopamine by DOPA decarboxylase,
and this dopamine converted to noradrenaline by
dopamine--hydroxylase. Finally, adrenaline is
produced via phenyl-ethanolamine-n-methyl
transferase.


39
HO
COOH
CH
2
-C-NH
2

H

HO
COOH
CH
2
-C-NH
2

H

HO
HO

CH
2
-CH
2
-NH
2



HO
HO
OH
C-CH
2
-NH
2

H

HO
TYROSINE DEHYDROXYPHENYLALANINE
(DOPA)
DOPAMINE
NORADRENALINE
HO
OH
C-CH
2
-NH-CH
3

H

HO
ADRENALINE
40
NEUROTRANSMITTERS.

Glycine, glutamate and aspartate function as
neurotransmitters predominantly as themselves.

Glutamate is also converted to -amino butyric
acid (GABA).

COOH

HOOC-CH
2
-CH
2
-CH-NH
2
HOOC-CH
2
-CH
2
-CH
2
-NH
2

GLUTAMATE DECARBOXYLASE

41
COOH
CH
2
-CH-NH
2
CH
2
-CH
2
-NH
2
HISTIDINE HISTAMINE

HO-CH
2
-CH
2
-N(CH
3
)
3
+
+ CH
3
-CO-CoA CH
3
-CO-O-CH
2
-CH
2
-N(CH
3
)
3
+
+ CoASH
CHOLINE ACETYLCoA ACETYLCHOLINE
CHOLINE ACETYL TRANSFERASE
HISTIDINE DECARBOXYLASE
Histidine is converted to histamine.
Choline is converted to acetylcholine.
42
HO
COOH
CH
2
-C-NH
2

H

HO
COOH
CH
2
-C-NH
2

H

HO
HO

CH
2
-CH
2
-NH
2



HO
HO
OH
C-CH
2
-NH
2

H

HO
TYROSINE DIHYDROXYPHENYLALANINE
(DOPA)
DOPAMINE
NORADRENALINE
Tyrosine is converted to dopamine and
noradrenaline.
43
COOH
CH
2
-CH-NH
2
COOH
CH
2
-CH-NH
2
HO

CH
2
-CH
2
-NH
2
HO
TRYPTOPHAN 5-HYDROXYTRYPTOPHAN
5-HYDROXYTRYPTAMINE
(SEROTONIN)
TRYPTOPHAN HYDROXYLASE
AROMATIC AMINO ACID
DECARBOXYLASE
Tryptophan is converted to serotonin.
44
AMINO ACID-RELATED DISEASES
Phenylketonuria, alkaptonuria, albinism, maple
syrup urine disease, ammonia toxicity,
Parkinsonism, schizophrenia, cystinuria, MSG,
urea cycle enzyme deficiency.
45
PHENYLKETONURIA (PKU)
This is an inborn error of metabolism with a
deficiency of phenylalanine hydroxylase. The
hydroxylation of phenylalanine is necessary in both
normal degradation and the conversion of
phenylalanine to tyrosine. The failure of this step
leads to accumulation and excretion of other
phenylalanine products, phenylpyruvate and
phenyllactate. The deficiency causes severe
mental retardation, minimal pigmentation, unusual
gait, stance and sitting posture, and an increased
frequency of epilepsy. The inability to synthesise
tyrosine is the primary underlying cause of the
cns problems, as dopamine and noradrenaline are
deficient. Failure to synthesise melanin is also a
direct result of tyrosine deficiency.
46
HO
COOH
CH
2
-C-NH
2

H

COOH
CH
2
-C-NH
2

H

COOH
CH
2
-CO
H

COOH
CH
2
-CHOH
H

MELANIN
DOPAMINE
NORADRENALINE
X
TYROSINE PHENYLALANINE
PHENYLPYRUVATE
PHENYLLACTATE
47
ALKAPTONURIA (BLACK URINE DISEASE)
Homogentisic acid is a breakdown product of both
phenylalanine and tyrosine and a deficiency of the
enzyme homogentisic acid oxidase that oxidises
this compound causes its accumulation and
excretion in the urine. Homogentisic acid auto-
oxidises and this gives the urine a very dark
color, hence the common name. This is a rare
condition, only about 1 in 1 million live births. It
is not a particularly life-threatening condition,
but can cause deposition of homogentisic acid into
cartilage tissues, and this can lead to severe
arthritis. There is no treatment other than to
relieve the pain.
48
MAPLE SYRUP URINE DISEASE (MSUD)
The metabolism of leucine, isoleucine and valine
initially removes the -amine group. This is
followed by decarboxylation of the resulting -
keto acid. This step is catalysed by branched-
chain keto acid decarboxylase, an enzyme located
inside the mitochondria. About 1 in 300 000 live
births show this defect, and the effects are for
them to pass the -keto acids in the urine, and
these give a maple syrup smell. If untreated it
can cause both physical and mental retardation.
The only treatment is a low protein diet.
49
CYSTINURIA
Different amino acids have different transport
systems. A defect of the cysteine transporter
system causes failure of reabsorption of cysteine
from the glomerular filtrate. The unabsorbed
cysteine oxidises to the dimer, cystine. Cystine is
much less soluble than cysteine and tends to
crystallise out, forming kidney stones. The
common preventative treatment is to restrict
intake of the cysteine precursor methionine,
although the stones can be treated with
ultrasound. Recently new drugs convert urinary
cystine to a more soluble form.
50
ALBINISM
This is reflection of the failure of the conversion
of tyrosine to DOPA via tyrosinase and the
subsequent failure to produce melanin. There does
not appear to be a total lack of the enzyme, but
rather a reduced capacity. The comparatively
small amounts of DOPA required for
neurotransmitter function can be produced, but
the much greater amounts required for
pigmentation are compromised. There is also
evidence that the enzyme that converts DOPA to
a melanin precursor quinone is deficient.
51
HEREDITARY HYPERAMMONEMIA
This condition reflects a deficiency in ornithine
transcarbamyolase, the key enzyme in condensing
carbamoyl phosphate onto ornithine and allowing
the urea cycle to clear excess ammonia as urea.
Excess ammonia will cause increased levels of
glutamate and glutamine, both of which will cause
cns disturbance. Arginine supplementation can help
to push the urea cycle faster as it is degraded to
urea and ornithine.

52
CHINESE RESTAURANT SYNDROME
This condition is a reflection of excessive levels
of glutamate in foods. The symptoms are a
mixture of headache, nausea, sweating, weakness,
facial flushes and tingling. Historically, it was
associated with chinese food as soy sauce contains
high levels of glutamate. It also underlies the
concerns about monosodium glutamate food
supplementation as similar effects have been
reported. Severe asthmatics may suffer
bronchospasm, but otherwise the effects reverse
rapidly.
53
PARKINSONISM (PARKINSONS DISEASE)
This is a common condition in the elderly, although
some young onset cases have been reported. It
reflects progressive loss of dopaminergic neurons
in the substantia nigra and locus ceruleus.
Dopamine is an inhibitory neurotransmitter in the
motor system, so uncontrollable tremor while
resting is a characteristic symptom. Frequent
cramping is also typical. It can be ameliorated
initially by DOPA and a monoamine oxidase (MAO)
inhibitor. The former is converted to dopamine,
the latter prevents it breakdown. The condition is
irreversible and progressive. The treatments also
have side effects on other CNS areas where
dopamine works.
54
SCHIZOPHRENIA
It has been suggested that increased dopamine in
the mesolimbic and mesocortical pathways may
contribute to schizophrenia. Some of the most
obvious evidence for this theory is from the
effects amphetamine and cocaine. These drugs
increase levels of dopamine in the brain and can
cause effects which resemble those present in
psychosis, particularly after large doses or
prolonged use. This is often referred to as
'amphetamine psychosis' or 'cocaine psychosis'
and may be virtually indistinguishable from
schizophrenia.

55
However, the acute effects of dopamine
stimulants include euphoria, alertness and over-
confidence; these symptoms are more reminiscent
of mania than schizophrenia. Other studies
showed that while some patients had over 90% of
their dopamine D2 receptors blocked by
antipsychotic drugs, they showed little reduction
in their psychoses. Depending on the study, there
was a spectrum of impact of dopaminergic
antagonists on schizophrenia.
56
THE END