Aspartate Transaminase - Mechanism

Mechanism

Aspartate transaminase, as with all transaminases, operates via dual substrate recognition; that is, it is able to recognize and selectively bind two amino acids (Asp and Glu) with different side-chains. In either case, the transaminase reaction consists of two similar half-reactions that constitute what is referred to as a ping-pong mechanism. In the first half-reaction, amino acid 1 (e.g., L-Asp) reacts with the enzyme-PLP complex to generate ketoacid 1 (oxaloacetate) and the modified enzyme-PMP. In the second half-reaction, ketoacid 2 (α-ketoglutarate) reacts with enzyme-PMP to produce amino acid 2 (L-Glu), regenerating the original enzyme-PLP in the process. Formation of a racemic product (D-Glu) is very rare.

The specific steps for the half-reaction of Enzyme-PLP + aspartate ⇌ Enzyme-PMP + oxaloacetate are as follows (see figure); the other half-reaction (not shown) proceeds in the reverse manner, with α-ketoglutarate as the substrate.

• Arg below, should indeed by Asp, as this is the mechanism for Aspartate, not Arginine. The structure of the nucleophilic Aspartate is correct.

1. Internal aldimine formation: First, the ε-amino group of Lys258 forms a Schiff base linkage with the aldehyde carbon to generate an internal aldimine.
2. Transaldimination: The internal aldimine then becomes an external aldimine when the ε-amino group of Lys258 is displaced by the amino group of aspartate. This transaldimination reaction occurs via a nucleophilic attack by the deprotonated amino group of Asp and proceeds through a tetrahedral intermediate. As this point, the carboxylate groups of Asp are stabilized by the guanidinium groups of the enzyme’s Arg386 and Arg 292 residues.
3. Quinonoid formation: The hydrogen attached to the a-carbon of Asp is then abstracted (Lys258 is thought to be the proton acceptor) to form a quinonoid intermediate.
4. Ketimine formation: The quinonoid is reprotonated, but now at the aldehyde carbon, to form the ketimine intermediate.
5. Ketimine hydrolysis: Finally, the ketimine is hydrolyzed to form PMP and oxaloacetate.

This mechanism is thought to have multiple partially rate-determining steps. However, it has been shown that the substrate binding step (transaldimination) drives the catalytic reaction forward.