Other AA products and Ammonia Disposal

Question 1

What are some fates of amino acids?

Answer 1

Protein synthesis
Glucose/glycogen
Energy (ATP)
Fatty acids/ketone bodies
Synthesis of nitrogen containing metabolites

Question 2

What are some nitrogen containing compounds?

Answer 2

Haem
Some neurotransmitters
Histamine
Creatine
Purine bases
Nitric oxide

Question 3

How is haem synthesised?

Answer 3

It dervies from succinyl-CoA and glycine starting out in the mitochondrion
2 molecules of δ-ala are combined to form porphobilinogen
4 of these units are combined to form uroporphyrinogen III, followed by various modifications – resulting in protoporphyrin IX, to which the Fe atom is attached
This goes out into the cytosol and then back into the mitochondria

Question 4

What are some mutations of haem enzymes?

Answer 4

Porphyrias are caused by accumulation of haem precursors
Acute porphyrias – abdominal pain, neurological and psychiatric features
Cutaneous porphyrias – photosensitivity, itching, blistering, increased hair growth
Some forms produce both types of symptoms

Treated by regular blood transfusions
Recent treatment involves injecting si RNAi to lower ALA and PBG

Question 5

Describe amines?

Answer 5

They are derived from amino acids
Examples:
Dopamine, noradrenalin, adrenaline, GABA, serotonin and histamine

Question 6

How are catecholamines synthesised?

Answer 6

Tyrosine is converted to L-DOPA using tyrosine hydrolase and a cofactor of tetrahydrobiopterin +O2
This can be turned into melanin or using aromatic amino acid decarboxylase be converted into dopamine
Dopamine can be further converted into noradrenaline and adrenaline

L-DOPA is used to treat Parkinson’s disease

Question 7

Describe nitric oxide?

Answer 7

Important nitrogen containing signalling molecule
It is a gas = rapid diffusion
Synthesised by three isoenzymes in endothelial cells (eNOS), neurons (nNOS) and macrophages (iNOS)

Question 8

How is nitric oxide (NO) derived?

Answer 8

L-Arginine + NADPH + O2 is converted to L-Hydroxyarginine

This undergoes the same reaction again to L-Citrulline to release NO

Question 9

What are some roles of nitric oxide?

Answer 9

Vasodilation – relaxation of smooth muscle (endothelial)
Control of blood pressure, control of renal filtration rate, penile erection, peristalsis
Nitroglycerin (treatment for angina) generates NO to dilate cardiac arteries
Sildenafil (Viagra) inhibits NO breakdown to improve erection
Neurotransmitter in central and peripheral nervous system (neurons)
Bacteriocidal effects of macrophages

Question 10

What can we do with excess amino acids?

Answer 10

Convert them to: Glucose, acetyl-CoA and ketone bodies

Get rid of it through making ammonia and then urea

Question 11

Why is ammonia converted? What is it converted into?

Answer 11

Ammonia is too toxic

Bacteria/fish - excrete ammonia directly into surrounding fluid
Reptiles/birds/desert animals - convert to uric acid (relatively insoluble in water)
Humans - convert to urea

Question 12

Give an overview of transamination?

Answer 12

The amino group is often transferred to an a-keto acid by transamination
The a-keto acid is often a-ketoglutarate, which is converted to glutamate
The a-ketoglutarate is regenerated in a second transamination reaction involving oxaloacetate

There are 3 stages

Question 13

What are the enzymes involved in transamination?

Answer 13

Transaminases

They contain the coenzyme pyridoxal-5´-phosphate (PLP)
PLP is derived from pyridoxine (vitamin B6)

PLP is covalently bound in the enzyme active site via a Schiff base (C=N) linkage to a Lys
The a-amino group of the amino acid substrate displaces Lys to form a new amino acid-PLP Schiff base
PLP goes to pyridoxamine-5’-phosphate (PMP)

Question 14

What is stage 1 of transamination?

Answer 14

Transimination
Amino acid to an a-keto acid

a-amino acid nucleophilically attacks the enzyme-PLP schiff base, which forms a geminal diamine intermediate before being converted to amino acid-PLP schiff base (aldimine) + enzyme

Question 15

What is stage 2 of transamination?

Answer 15

Tautomerization
Aldimine is tautomerized to ketimine via a resonance stablised intermediate
Ketimine is nucleophilically attacked by a OH-, forming a resonance stabalised intermediate, before forming carbinolamine

Pyridinium nitrogen is an electron sink – ‘spreads’ excess electrons across several conjugated pi bonds - within the intermediate

Question 16

What is stage 3 of transamination?

Answer 16

Hydrolysis

Carbinolamine is hydrolysed into Pyridoxamine phosphate (PMP) - enzyme and and a-keto acid

Question 17

What happens to the PMP after transamination?

Answer 17

It needs to be converted back into enzyme-PLP schiff base to complete the amino transferases catalytic cycle

1. PMP reacts with an α-keto acid to form a Schiff base
2. The α-keto acid–PMP Schiff base tautomerizes to form an amino acid–PLP Schiff base
3. The amino group of the active site Lys attacks the amino acid-PLP schif base to form enzyme-PLP schiff base and an amino acid

Question 18

Can all amino acids be transaminated?

Answer 18

No lysine can’t

Question 19

What are transaminases used for?

Answer 19

Transaminase levels in serum are good diagnostic indicators

ALT (alanine transaminase) - found in liver
Aspartate transaminase (AST) - found widely

Raised serum levels of ALT & AST found in hepatocyte damage
Therefore helpful in determining the cause of liver damage
Can also be used to confirm diagnosis of heart attacks

Question 20

Describe PLP?

Answer 20

This is a cofactor for enzymes that act on amino acids
It is derived from vitamin B6
PLP is also used to create physiologically active amines by decarboxylation of amino acids

Required for:
transamination and synthesis of non-essential amino acids
decarboxylation reactions required for neurotransmitter synthesis (e.g. GABA)
haem synthesis
some aspects of energy metabolism & lipid synthesis (e.g. Ceramide)

Question 21

What does lack of B6 lead to?

Answer 21

Anaemia - lack of haem for haemoglobin
Neurological symptoms (depression, confusion, seizures) - lack of neurotransmitter & lipid synthesis
Poor growth, skin lesions, poor immune responses - lack of protein synthesis

Question 22

What are some general examples of PLP being used as a cofactor?

Answer 22

Cleavage of bond a (C-H) results in transamination or racemisation (interconversion between D/L amino acids)
Cleavage of bond b (C-COOH) results in decarboxylation e.g. Glu -> GABA
Haem is synthesised from glycine and succinyl-CoA
Cleavage of bond c results in removal of the sidechain e.g. Interconversion between serine and glycine

Question 23

What is a specific example of PLP being used?

Answer 23

The amino acid-PLP Schiff base (aldimine) - this has several possible fates

Depending upon which of bonds a, b or c is broken - in relation to the pi-orbital system in the PLP:
Which bond is broken depends upon the orientation of the groups attached to Ca
The bond that lies in a plane perpendicular to the PLP p-orbitals is the most liable
Electrons from the broken bond can delocalise across PLP - stereoelectronic effect

Question 24

What are the stages of disposal of ammonia in animals?

Answer 24

3 step process:
Transamination -transfer of amino group from amino acid to a-ketoglutarate forming glutamate (in most tissues)
Deamination - release of ammonia from glutamate (mainly in the liver)
Urea Synthesis - urea cycle (in the liver)

Question 25

What is also involved in the transamination reactions, asid from formation of glutamine?

Answer 25

The Glucose-alanine cycle

This allows alanine to transfer ammonia to the liver

Question 26

Describe oxidative deamination of glutamate?

Answer 26

Glutamate is deaminated to a-ketoglutarate by glutamate dehydrogenase - releasing ammonium
Unusual enzyme as it can use both NAD and NADP as a cofacor
Glutamate is transported from the cystol into the mitochondria as GDH is a mitochondrial enzyme

Stimulates ATP synthesis as a-KG is a citric acid cycle intermediate
Equilibrium of the reaction lies towards ammonia release, not ammonia removal

Question 27

What is a disease of glutamate dehydrogenase?

Answer 27

Hyperammonemia - elevated levels of ammonia in the blood

This is a result of mutations in GDH = decreased sensitivity to GTP inhibition, resulting in increased GDH activity

Question 28

Give an overview of urea?

Answer 28

Urea consists of 2 amino groups, one from ammonia, one from aspartate plus one C from bicarbonate at the ‘expense’ of 4 phosphate bonds
The energy is worth using, as more ATP than this can be obtained by the entry of the C-skeletons into the TCA cycle

Urea is synthesised in the liver - during the urea cycle

Question 29

What are the enzymes involved in the urea cycle?

Answer 29

Carbamoyl phosphate   synthetase
Ornithine transcarbamoylase
Argininosuccinate synthetase
Argininosuccinase
Arginase

Question 30

What is stage 1 of the urea cycle?

Answer 30

Carbamoyl phosphate synthetase (CPS1 - the mitochondrial isoenzyme) takes the ammonia produced in deamination
2ATP + HCO3- + NH3 -> Carbamoyl phosphate 2ADP + Pi

In the mitochondria

Question 31

Describe CPS specifically in E.Coli?

Answer 31

A multi-enzyme complex - combines NH3 release from glutamine with carbamoyl phosphate synthesis

NH3 travels down a tunnel to react with ATP and HCO3- to form carbamate
Carbamate follows the tunnel to the final active site where carbamoyl phosphate is synthesised
This is known as channelling - which protects intermediates from degradation (very reactive) and allows relatively high concentrations of NH3

Question 32

What is stage 2 of the urea cycle?

Answer 32

Ornithine transcarbamoylase converts carbamoyl phosphate and Ornithine into citrulline
This is a non standard a-amino acid that don’t occur in proteins

In the mitochondria
Citrulline is now transported out the mitochondria into the cytosol

Question 33

What is stage 3 of the urea cycle?

Answer 33

Using argininosuccinate synthestase and ATP
Aspartate is added to citrulline to form Argininosuccinate
It forms a citrullyl-AMP intermediate
This acquires the 2nd urea nitrogen atom

Question 34

What is stage 4 of the urea cycle?

Answer 34

Argininosuccinase converts Argininosuccinate into Arginine and Fumarate
Arginine is ureas immediate precursor
Fumarate is converted into malate then oxaloacetate to be used in gluconeogenesis

Question 35

What is stage 5 of the urea cycle?

Answer 35

Arginase converts Arginine into Ornithine and urea
Urea is released
Ornithine is regenerated to be reused in the cycle

Question 36

How is the urea cycle regulated?

Answer 36

CPS1 is the rate limiting step
CPS1 is allosterically activated by N-acetlyglutamate

Amino acids are broken down and glutamate concentration increases due to transamination
Increased glutamate = N-acetlyglutamate synthesis