Aminotransferase Mechanism Flashcards
What type of kinetics is aminotransferase an example of?
Bi-Bi ping-pong kinetics.
How is glutamate synthesised?
From α-KG and ammonia de novo.
How can other chiral amino acids be synthesised?
By transamination- stereospecific transfer of the amino group in glutamate to the α carbon.
What are the two main stages involved in transamination?
- Conversion of an amino acid to α-keto acid.
2. Conversion of an α-keto acid to an amino acid.
What are the 3 steps involved in each of the stages of transamination?
- Transimination
- Tautomerization
- Hydrolysis
What is an aldimine?
A schiff base that has formed between an imine and aldehyde.
What is a ketimine?
A schiff base that has formed between an imine and a ketone.
How is PLP attached to the aminotransferase?
Covalently attached to the ɛ-amino group of a lys residue in the active site.
What allows PLP to act as a mobile electron sink?
Its stable tautomeric forms.
Describe transimination (1).
Nucleophilic attack of the enzyme-PLP schiff base, forming aldimine and releasing the lys residue.
Describe tautomerization (2).
Lys acts as a general base and abstracts a proton, forming a carbanion- stabilised by PLP.
Amino acid-PLP schiff base tautomerises to an α-keto acid-PMP schiff base via a resonance stabilised quinonoid intermediate.
Decribe hydrolysis (3).
α-keto acid-PMP schiff base is hydrolysed to give PMP and α-keto acid 1.
Describe transimination (1’).
Nucleophilic attack of the enzyme-PMP schiff base to form ketimine and release the lys residue.
Describe tautomerisation (2’).
α-keto acid-PMP schiff base tautomerises to form an amino acid-PLP schiff base.
Describe hydrolysis (3’).
ɛ-amino group of lys attacks the amino acid-PLP schiff base, regenerating the enzyme-PLP schiff base and forming amino acid 2.
What does resonance stabilisation depend on?
The planarity of the quinonoid intermediate
- molecule must be planar to allow π-orbital overlap to form an orbital that covers the whole molecule.
What is the stereospecificity is controlled by?
The configuration of the intermediate, which depends on how molecules are held in the active site.
How is the carboxylate of the α carbon of the amino acid held in the active site?
By electrostatic interaction with an Arg residue.
Which amino acids do not have a 2S configuration?
Gly and Cys.
What are other reactions of the α carbon are catalysed by PLP-dependent enzymes?
- transamination
- racemisation
- decarboxylation
- aldolase reactions (reverse of aldol condensation)
What reactions of the β or γ carbon are catalysed by PLP-dependent enzymes?
- elimination
- replacement
What is the amino acid usually used in transamination and what is the α keto acid produced?
glutamate, producing α-KG.
