Other AA products and Ammonia Disposal Flashcards
What are some fates of amino acids?
Protein synthesis Glucose/glycogen Energy (ATP) Fatty acids/ketone bodies Synthesis of nitrogen containing metabolites
What are some nitrogen containing compounds?
Haem Some neurotransmitters Histamine Creatine Purine bases Nitric oxide
How is haem synthesised?
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
What are some mutations of haem enzymes?
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
Describe amines?
They are derived from amino acids
Examples:
Dopamine, noradrenalin, adrenaline, GABA, serotonin and histamine
How are catecholamines synthesised?
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
Describe nitric oxide?
Important nitrogen containing signalling molecule
It is a gas = rapid diffusion
Synthesised by three isoenzymes in endothelial cells (eNOS), neurons (nNOS) and macrophages (iNOS)
How is nitric oxide (NO) derived?
L-Arginine + NADPH + O2 is converted to L-Hydroxyarginine
This undergoes the same reaction again to L-Citrulline to release NO
What are some roles of nitric oxide?
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
What can we do with excess amino acids?
Convert them to: Glucose, acetyl-CoA and ketone bodies
Get rid of it through making ammonia and then urea
Why is ammonia converted? What is it converted into?
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
Give an overview of transamination?
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
What are the enzymes involved in transamination?
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)
What is stage 1 of transamination?
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
What is stage 2 of transamination?
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
What is stage 3 of transamination?
Hydrolysis
Carbinolamine is hydrolysed into Pyridoxamine phosphate (PMP) - enzyme and and a-keto acid
What happens to the PMP after transamination?
It needs to be converted back into enzyme-PLP schiff base to complete the amino transferases catalytic cycle
- PMP reacts with an α-keto acid to form a Schiff base
- The α-keto acid–PMP Schiff base tautomerizes to form an amino acid–PLP Schiff base
- 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
Can all amino acids be transaminated?
No lysine can’t
What are transaminases used for?
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
Describe PLP?
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)
What does lack of B6 lead to?
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
What are some general examples of PLP being used as a cofactor?
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
What is a specific example of PLP being used?
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
What are the stages of disposal of ammonia in animals?
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)
What is also involved in the transamination reactions, asid from formation of glutamine?
The Glucose-alanine cycle
This allows alanine to transfer ammonia to the liver
Describe oxidative deamination of glutamate?
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
What is a disease of glutamate dehydrogenase?
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
Give an overview of urea?
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
What are the enzymes involved in the urea cycle?
Carbamoyl phosphate synthetase Ornithine transcarbamoylase Argininosuccinate synthetase Argininosuccinase Arginase
What is stage 1 of the urea cycle?
Carbamoyl phosphate synthetase (CPS1 - the mitochondrial isoenzyme) takes the ammonia produced in deamination
2ATP + HCO3- + NH3 -> Carbamoyl phosphate 2ADP + Pi
In the mitochondria
Describe CPS specifically in E.Coli?
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
What is stage 2 of the urea cycle?
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
What is stage 3 of the urea cycle?
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
What is stage 4 of the urea cycle?
Argininosuccinase converts Argininosuccinate into Arginine and Fumarate
Arginine is ureas immediate precursor
Fumarate is converted into malate then oxaloacetate to be used in gluconeogenesis
What is stage 5 of the urea cycle?
Arginase converts Arginine into Ornithine and urea
Urea is released
Ornithine is regenerated to be reused in the cycle
How is the urea cycle regulated?
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
