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Search The CSA
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Catalytic Site Atlas

CSA LITERATURE entry for 2aat

E.C. nameaspartate transaminase
SpeciesEscherichia coli (Bacteria)
E.C. Number (IntEnz) 2.6.1.1
CSA Homologues of 2aatThere are 16 Homologs
CSA Entries With UniProtID P00509
CSA Entries With EC Number 2.6.1.1
PDBe Entry 2aat
PDBSum Entry 2aat
MACiE Entry 2aat

Literature Report

IntroductionAspartate aminotransferase is a pyridoxal 5'-phosphate(PLP)-dependent enzymes that catalyses the reversible transamination of L-amino acids, aspartate and glutamate and the corresponding 2-oxo acids, oxalacetate and 2-oxoglutarate. All members of the PLP-dependent enzyme families use the same protein scaffold to catalyse quite diverse reactions.
MechansimIn the enzyme, PLP is anchored to the protein at Lys258 with its pyridoxal moiety in the reactive bipolar ionic form. In the first step, the cofactor PLP is transferred from Lys 258 to the aspartate alpha amino group. The alpha-amino group of the aspartate attacks the PLP C4' from the front side in a direction perpendicular to the plane of the pyridine ring. The attraction of opposite charges on the substrate N atom and O3' of the coenzyme brings about a 90 degrees rotation of the 2 C-N bonds around C4-C4' which brings Lys258 behind the plane of the pyridine ring and hence Lys258 can be released. Trp 140 sterically constraining the PLP to allow the attack of aspartate.
The deprotonation of the alpha-carbon of aspartate gives rise to the quinonoid intermediate, which is stabilised by the electron link of the protonated pyridine ring. Asp223 facilitates the deprotonation by stabilising the positive charge at N1 of PLP with a salt bridge and hence enhancing the electron withdrawing capacity of the amino acid substrate. Lys 258 acts as a base here.
Lys258 then protonates C4' from the si side, giving rise to keimine intermediate. It then deprotonates a water molecule to allow its nucleophilic attack on the alpha-carbon. The tetrahedral intermediate dissociates into PMP and oxo-acid product. Subsequent reversal of the reaction steps described occurs except with the carboxyl group of the alpha keto-glutarate acting as the nucleophile leading to the formation of glutamate and the completion of the reaction cycle.
Reaction

Catalytic Sites for 2aat

Annotated By Reference To The Literature - Site 1 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
TrpA142130macie:sideChainIt sterically constrains the PLP cofactor in order to make nucleophilic attack by the aspartate on PLP more favourable.
AspA223211macie:sideChainIt forms a salt bridge with N1 of pyridine ring and enhance the electron withdrawing capacity of the pyridine ring to facilitate the removal of the alpha-proton of the amino acid substrate.
AlaA258246macie:sideChainIt binds to PLP cofactor. It acts as acid base for the conversion of the aldimine to the ketimine forms of the PLP-aspartate intermediate. It also deprotonates a water molecule for the nucleophilic attack on the alpha-carbon to form the 2-oxoacid product from the amino acid.

Literature References

Notes:
Kirsch JF
Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure.
J Mol Biol 1984 174 497-525
PubMed: 6143829
Smith DL
2.8-A-resolution crystal structure of an active-site mutant of aspartate aminotransferase from Escherichia coli.
Biochemistry 1989 28 8161-8167
PubMed: 2513875
Yano T
Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate.
Biochemistry 1992 31 5878-5887
PubMed: 1610831
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