Download Amino Acids Objectives

Amino Acids: Parts 1 and 2
1. Which amino acids are the essential and which are nonessential? Which
nonessential amino acids are derived directly from an essential amino acid? Which
amino acids are conditionally essential and why?
Cysteine is made from methionine. If tyrosine is not adequate in the diet, large
amounts of phenylalanine are required in order to produce it. Arginine and histidine are
not synthesized in adequate quantities only when they are supporting growth in young
animals.
2. How are amino acids absorbed in small intestine?
Dietary protein is denatured under acidic conditions and partially digested by
pepsin in the stomach. Broken down in the small intestine by trypsin, chymotrypsin,
elastase, aminopeptidases, and carboxypeptideases. Didpeptidase (enterocytes) digests
proteins and peptides. Amino acids are absorbed by these enterocytes on the luminal side
by multiple carriers. Dipeptides use H+-dependent carriers. Separate basolateral carriers
transport amino acids into the blood.
Types of carriers that bring amino acids into the enterocytes:
1.)Na+-dependent amino acid transport systems with overlapping amino acid
specificities
2.)Na+ and amino acids at high luminal concentrations are co-transported due to a
concentration gradient into cells
3.)The ATP-dependent Na+/K+ pump exchanges the intracellular Na+ for
extracellular K+, reducing Na+ levels to maintain the extracellular Na+
concentration gradient to drive transport
4.)Small peptides are transported by a proton (H+) driven transporter and
hydrolyzed by intracellular peptidases.
5.)Amino acids in blood and extracellular fluids are transported by at least 7 ATPrequiring active transport systems with overlapping amino acid specificities
3. What molecules serve as carriers of nitrogen?
Urea – ureotelic organisms; mammals. Nontoxic, highly water soluble (up to 10M), used
only for excretion of excess nitrogen. Formed from ammonia and bicarbonate.
Tranfers nitrogen in blood for excretion in the urine.
Purines – purinotelic organisms; bats, birds, reptiles. Excreted in feces. Minimizes use of
water. Great fertilizer.
Ammonia – most invertebrates. Relatively small amounts excreted normally in urine by
humans. Fish excrete ammonia through their gills. Small organisms can excrete
ammonia directly into their environment.
Glutamine – relatively nontoxic. Formed from glutamate and ammonia. Water soluble.
An amino group donor in a number of pathways (synthesis of a lot of purines).
Carrier of nitrogen to liver from muscles for synthesis of urea.
Glu = Glutamate, Gln = Glutamine
Alanine – nontoxic. Formed from pyruvate and an amino acid. Used for inter-organ
transfer of nitrogen (glucose-alanine cycle).
4. What properties of urea make it well suited as a carrier of nitrogen?
It’s nontoxic, highly water soluble, and used only for the excretion of nitrogen (a
“metabolic dead end”).
5. Explain how transaminases and glutamate dehydrogenase play a central role in
nitrogen metabolism.
Transaminases transfer amino groups from amino acids (mainly alanine and
glutamine) to α-ketoglutarate to make glutamate. Glutamate dehydrogenase then uses
NAD+ to release ammonia for urea synthesis and be regenerated to α-ketoglutarate
(oxidative deamination of glutamate). Glutamate dehydrogenase can use NADP+, but the
predominant form used in vivo is NADPH toward glutamate and NAD+ toward αketoglutarate. It is confined to the mitochondrial matrix.
6. Under what conditions are ammonium ions excreted in relatively high
concentrations in the urine? What enzyme plays an important role under these
conditions?
Metabolic acidosis. Glutaminase plays an important role under these conditions.
(Note: urea production is suppressed. Nitrogen is being directly excreted as NH4+.)
Glutamine + H2O → Glutamate + NH4+
7. Explain how the glucose alanine cycle operates in inter-organ metabolism. Why is
this cycle important?
It’s important to provide for a system to transport nitrogen generated by
metabolism of amino acids from one organ to another in a nontoxic form. It also produces
pyruvate for production of glucose by gluconeogenesis in the liver. If you start in muscle,
pyruvate (from metabolism of glucose) can be converted to alanine by transferring an
amino group from some other amino acid to pyruvate by alanine aminotransferase
(glutamate pyruvate amino transferase, GPT). This can be transferred in the blood to the
liver, where it’s either directly deaminated to give ammonia, or it’s transferred to another
amino acid, which goes to glutamate, which is eventually released as ammonia. One
nitrogen from alanine is recovered to produce ammonia, which goes to production of urea.
What is left from alanine produces pyruvate. Pyruvate is a gluconeogenic precursor, so
glucose can be formed.
Liver
Muscle
8. Explain how pyridoxal phosphate functions in amino group transfer by
transaminases.
Pyridoxal phosphate is an electrophile for covalent catalysis in amino group
transfer by transaminases.
9. How are the reactions of the urea cycle compartmentalized inside cells?
In the mitochondria –
First: activated 5-10 fold by N-acetylglutamate
Second: Ornithine is made in the cytosol. Ornithine and citrulline require a
membrane carrier to enter or exit the mitochondria
Citrulline is then transported to the cytosol where the next three reactions occur.
In the cytosol –
Third: Citrulline is used to make argininosuccinate
Fourth: Arginine and fumarate are formed from arginosuccinate
Finally: Arginase releases urea from arginine. Arginase is present in the liver, but
absent in the kidney (which is a major site of arginine synthesis).
!!SUMMARY!!
10. Explain the role of the shuttles for ornithine/citrulline and malate/aspartate in
the urea cycle.
The malate/aspartate shuttle moves aspartate, glutamate, and α-ketoglutarate
across the mitochondrial membrane by converting malate to oxaloacetate, and that to
aspartate. Malate can be transported into the mitochondria as an antiport with αketoglutarate. Aspartate can be transported into the cytosol as an antiport with glutamate.
Ornithine/citrulline is an antiport that carries citrulline and ornithine across the
mitochondrial membrane. This is important because citrulline is being produced in the
mitochondria during the second step of the urea cycle but reacts in the third step in the
cytosol. Ornithine, on the other hand, is one of the final products of the urea acid cycle
and can be transported back to the mitochondria to function as a reactant in the second
reaction to make more citrulline. (See above image.)
11. How are the urea cycle and the TCA cycle linked?
The urea cycle, through arginosuccinate lyase, produces fumarate. Fumarate can
be combined with water via fumarase to produce malate, which is an intermediate in the
TCA cycle.
Aspartate can give rise to oxaloacetate through the action of a transaminase. So
you can get aspartate from oxaloacetate in the TCA cycle, and then the aspartate can be
used in the urea cycle to form arginosuccinate.
12. How is the urea cycle regulated?
Long term regulation – elevated synthesis of urea cycle enzymes due to increased
metabolism of amino acids, either from diet or from protein turnover (starvation).
If in the case of a high protein diet, the carbon skeletons derived from amino acids
in the diet are oxidized or converted to fat or glycogen. The nitrogen is eliminated
through the synthesis of urea. Enzymes of the urea cycle are induced up to 20 fold
over a low protein diet. In the case of starvation, the carbon skeletons derived
from the breaking down of muscle (protein catabolism) are oxidized and nitrogen
is used for urea. Enzymes of urea cycle are induced.
Short term regulation – Carbamoyl phosphate synthetase I is allosterically activated 5-10
fold by N-acetylglutamate. Synthesis of N-acetylglutamate depends on the
availability of Acetyl-CoA and glutamate. Arginine activates N-acetylglutamate
synthase. The precursors to N-acetylglutamate and arginine are signals of high
levels of amino acids whose metabolism will generate ammonia.
Acetyl-CoA + glutamate → N-acetylglutamate + CoA
13. Explain why ammonia is toxic. Explain the elements of inter-organ metabolism
that contribute to the self-perpetuating nature of ammonia intoxication.
It alters the production of neurotransmitters in the brain and reduces intracellular
ATP. It causes swelling of the brain to the point that it squeezes out the bottom of the
brain (liver failure). (Remember, it is very membrane permeable; it can cross the
blood:brain barrier.)
Elevated NH4+ concentration due to liver failure stimulates glucagon release in
the pancreas, which stimulates gluconeogenesis from amino acids in the kidney, resulting
in more NH4+. This ammonium stimulates more glucagon release, which stimulates
release of more glucose (gluconeogenesis), and glucose promotes insulin release from the
pancreas. This insulin stimulates uptake of branched chain amino acids by muscle, where
they are used as oxidizable substrates. Their metabolism releases even more NH4+.
This elevated NH4+ concentration in the brain stimulates glutamine synthesis,
which in turn promotes tryptophan transport, which is already increased through the
transporter for neutral amino acids because their serum levels decline as they are
metabolized in muscle. Elevated tryptophan increases the synthesis of 5-OH-tryptamine,
which is a reactive oxygen species adding oxidative stress in the brain.
14. What TCA cycle intermediate is depleted in the brain during ammonia
intoxication and how does this happen? What amino acids are depleted in the
brain? What are the consequences of depletion of these metabolites for the brain?
α-ketoglutarate is depleted in the brain. Glutamate is also depleted. There is not as
much α-ketoglutarate the flow through the TCA cycle for ATP production, there is not as
much glutamate for the synthesis of γ-amino butyric acid (GABA), ATP utilization is
increased, and there is increased oxidative stress due to the presence of reactive oxidative
species.
15. How is ammonia intoxication treated and how do these treatments work?
Diet – low protein, high carbohydrate diet reduces NH4+ production
Levulose – bacteria in the colon metabolize levulose to acidic products, which promotes
ammonia excretion in the feces as NH4+, which is less readily absorbed than NH3
in the gut.
Antibiotics – to kill intestinal ammonia-producing bacteria
Sodium benzoate and sodium phenylbutyrate – form covalent products with glycine
(hippurate) and glutamine (phenylacetylglutamine). These compounds promote
nitrogen excretion in the feces.
16. Explain the distinction between a ketogenic vs. a gluconeogenic fate of an amino
acid. Which amino acids are exclusively ketogenic and to what are they ultimately
metabolized?
Ketogenic amino acids yield precursors to fatty acids or ketone bodies. They are
metabolized to acetyl-CoA and acetoacetyl-CoA. Leucine and lysine are exclusively
ketogenic amino acids.
Glucogenic amino acids yield precursors that can be used of gluconeogenesis.
They are metabolized to pyruvate, α-ketoglutarate, succinyl-CoA, fumarate, or
oxaloacetate.
17. To what TCA cycle intermediates are individual amino acids metabolized and
where do these metabolites enter the TCA cycle?
18. What metabolites accumulate in individuals with a defect in the branched chain
keto acid dehydrogenase? What amino acids are metabolized by the pathway
involving this enzyme?
Α-ketoisocaproate, α-keto-β-methylvalerate, and α-ketoisovalerate. Leucine,
isoleucine, and valine are metabolized by this pathway. Causes severe mental retardation.
It’s a rare, recessive mutation isolated in Asian and Caucasian (Mennonites) communities.
19. What metabolites accumulate in alkaptonuria and what enzyme is defective in
this disease? How are the clinical manifestations of this disorder related to the
accumulation of this metabolite?
Homogentisate oxidase is defective, causing homogentisate to accumulate.
Patients have a damage to joints, calcifications in the urinary tract and cardiovascular
system, and a reddish tint (ochre) to the skin. Urine also turns black upon standing at
room temperature for 24 hours. All of this is due to accumulation of homogentisate.
20. How is S-adenosyl methionine synthesized?
Methionine and ATP are added via methionine adenosyl-transferase to produce sadenosylmethionine (SAM).
21. How is methionine regenerated from homocysteine? What coenzyme is involved
in this reaction? What other enzyme catalyzed reaction in mammals utilizes this
coenzyme?
N5-CH3 THF is converted to THF via methionine synthase. B12 is required for
this reaction.
The interconversion of methylmalonyl-CoA and succinyl-CoA utilizes
methylmalonyl-CoA mutase, which also requires coenzyme B12. B12 dependent
enzymes catalyze intramolecular or intermolecular group transfer.
22. What enzyme is defective in individuals with homocystinuria? What metabolite
accumulates? How is this disease usually treated?
Cysathionine β-synthase, which is a vitamin B6 dependent enzyme.
Homocysteine accumulates. Two kinds of treatment: vitamin responsive and
unresponsive. Vitamin responsive can be treated with pyridoxine (vitamin B6), folic acid,
and vitamin B12. Vitamin unresponsive is treated with a methionine restricted diet
supplemented with cystine and betaine (promotes remethylation of homocysteine to
methionine).
23. What is the nature of the defect in cystinosis and how is this disorder treated
clinically?
There is a defect in a cystine transporter (cystinosin) in the lysosomes, so cystine
accumulates there. There is often a deposition of cystine crystals in tissues (like kidneys
and eyes). Treated with cysteamine, which reacts with cystine in the lysosomes to form
cysteine-cysteamine; this can then be transported out of the lysosomes through a
transporter for lysine.
24. How are homocysteine thiolactone and S-nitrosohomocysteine formed?
Homocysteine is formed from methionine through S-adenosylmethionine.
Homocysteine thiolactone is made from homocysteine by methionyl-tRNA
synthetase. S-nitrosohomocysteine is formed from homocysteine and NO. Both of these
react with proteins and are cytotoxic. (They react with protein amino groups, rendering
the modified protein immunogenic.)
25. Which amino acids can be made from intermediates of a central pathway and
what intermediate gives rise to which amino acids?
From intermediates of a central pathway:
Phosphoglycerate → Serine
Phosphoglycerate → Serine → Glycine
Citrulline → Argininosuccinate → Arginine (urea cycle)
From other amino acids:
Phenylalanine → Tyrosine
Glutamate → Proline
Methionine →Cysteine
26. Explain the role of the small intestine and kidney in the synthesis of arginine.
Small intestine – citrulline is synthesized in the enterocytes; involves the synthesis of
ornithine by transamination of glutamate γ-semialdehyde followed by reaction
with carboamoyl phosphate (beginning of urea cycle). Citrulline is then carried by
the circulation to the kidney.
Kidney – citrulline taken up by kidney from circulation. Citrulline is combined with
aspartate and ATP to produce argininosuccinate. The kidneys do not have arginase to
complete the urea acid cycle, however; they have argininosuccinate lyase, which
produces fumarate and arginine.
This whole pathway is called the intestinal-renal axis.
27. What reaction is catalyzed by phenylalanine hydroxylase and what is the role of
tetrahydrobiopterin in this reaction. What is phenylketonuria?
Phenylalanine → Tyrosine is catalyzed by phenylalanine hydroxylase.
Tetrahydrobiopterin is the required coenzyme and acts as the immediate reductant that
gives up its H. It is then regenerated by NADH + H+.
Phenylketonuria is when phenylaline hydroxylase is defective, or occasionally the
dihydrobiopterin cofactor. If left untreated it can result in mental retardation. Affected
individuals are placed on a phenylalanine restricted, tyrosine supplemented diet; this must
be maintained until development is complete and while pregnant.
28. How is nitric oxide made? What are some of the functions of this short-lived
molecule?
Arginine + O2 via nitric oxide synthase produces citrulline. There are 3 isozymes,
each with different functions:
iNOS or NOSI – inducible form – immune and inflammatory responses
nNOS or NOSII – neurotransmitter in GI tract, in penile erection, sphincter relaxation
eNOS or NOSIII – endothelial cells – blood flow, blood pressure platelet activation
EXTRAS:
METABOLISM OF AMINO ACIDS
Amino acids metabolized to pyruvate:
Glycine cleavage system – reversible step in vitro, but in vivo it functions primarily in
serine and glycine catabolism and N5N10CH2THF production.
Serine – major route of serine catabolism and the principal source of glycine:
– relatively minor route of serine catabolism:
Threonine:
Valine, Isoleucine, and Leucine – all are changed to an α-keto-ate via a transaminase and
then lose CO2 and add CoASH via a branched chain keto acid dehydrogenase complex.
(Don’t worry about structures or intermediates.)
Lysine – exclusively ketogenic. Forms sarccharopine using α-ketoglutarate, then gives
rise partly to 2-amino-adipate, which is converted to acetoacetyl-CoA.
Phenylalanine and Tyrosine – both are ketogenic
Cysteine:
SYNTHESIS OF AMINO ACIDS
Asparagine – Glutamine is the donor in animal cells, NH3 is the N donor in bacteria and
plants:
Glutamine:
Arginine – covered in the objectives. (Split between the small intestine and kidneys.)
Tyrosine – covered in the objectives. (The role of phenylalanine hydroxylase.)
Cysteine:
Proline – from glutamate
Serine – derived from a glycolytic intermediate, 3-phosphoglycerate
Glycine – source of most of the glycine required for other biosynthetic processes,
including protein synthesis and pyrimidine synthesis. Also contributes to the one
carbon pool (N5N10CH2THF)