Aminotransferase Mechanism

Question 1

What type of kinetics is aminotransferase an example of?

Answer 1

Bi-Bi ping-pong kinetics.

Question 2

How is glutamate synthesised?

Answer 2

From α-KG and ammonia de novo.

Question 3

How can other chiral amino acids be synthesised?

Answer 3

By transamination- stereospecific transfer of the amino group in glutamate to the α carbon.

Question 4

What are the two main stages involved in transamination?

Answer 4

1. Conversion of an amino acid to α-keto acid.

2. Conversion of an α-keto acid to an amino acid.

Question 5

What are the 3 steps involved in each of the stages of transamination?

Answer 5

1. Transimination
2. Tautomerization
3. Hydrolysis

Question 6

What is an aldimine?

Answer 6

A schiff base that has formed between an imine and aldehyde.

Question 7

What is a ketimine?

Answer 7

A schiff base that has formed between an imine and a ketone.

Question 8

How is PLP attached to the aminotransferase?

Answer 8

Covalently attached to the ɛ-amino group of a lys residue in the active site.

Question 9

What allows PLP to act as a mobile electron sink?

Answer 9

Its stable tautomeric forms.

Question 10

Describe transimination (1).

Answer 10

Nucleophilic attack of the enzyme-PLP schiff base, forming aldimine and releasing the lys residue.

Question 11

Describe tautomerization (2).

Answer 11

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.

Question 12

Decribe hydrolysis (3).

Answer 12

α-keto acid-PMP schiff base is hydrolysed to give PMP and α-keto acid 1.

Question 13

Describe transimination (1’).

Answer 13

Nucleophilic attack of the enzyme-PMP schiff base to form ketimine and release the lys residue.

Question 14

Describe tautomerisation (2’).

Answer 14

α-keto acid-PMP schiff base tautomerises to form an amino acid-PLP schiff base.

Question 15

Describe hydrolysis (3’).

Answer 15

ɛ-amino group of lys attacks the amino acid-PLP schiff base, regenerating the enzyme-PLP schiff base and forming amino acid 2.

Question 16

What does resonance stabilisation depend on?

Answer 16

The planarity of the quinonoid intermediate

- molecule must be planar to allow π-orbital overlap to form an orbital that covers the whole molecule.

Question 17

What is the stereospecificity is controlled by?

Answer 17

The configuration of the intermediate, which depends on how molecules are held in the active site.

Question 18

How is the carboxylate of the α carbon of the amino acid held in the active site?

Answer 18

By electrostatic interaction with an Arg residue.

Question 19

Which amino acids do not have a 2S configuration?

Answer 19

Gly and Cys.

Question 20

What are other reactions of the α carbon are catalysed by PLP-dependent enzymes?

Answer 20

• transamination
• racemisation
• decarboxylation
• aldolase reactions (reverse of aldol condensation)

Question 21

What reactions of the β or γ carbon are catalysed by PLP-dependent enzymes?

Answer 21

• elimination

- replacement

Question 22

What is the amino acid usually used in transamination and what is the α keto acid produced?

Answer 22

glutamate, producing α-KG.