PDBsum entry 1map

Aminotransferase

PDB id: 1map

Contents

Protein chain

401 a.a. *

Ligands

KET

Waters ×306

* Residue conservation analysis

PDB id:

1map

Name:

Aminotransferase

Title:

Crystal structures of true enzymatic reaction intermediates: aspartate and glutamate ketimines in aspartate aminotransferase

Structure:

Aspartate aminotransferase. Chain: a. Engineered: yes

Source:

Gallus gallus. Chicken. Organism_taxid: 9031. Organ: heart. Expressed in: unidentified. Expression_system_taxid: 32644

Biol. unit:

Dimer (from PQS)

Resolution:

2.40Å R-factor: 0.169

Authors:

V.N.Malashkevich,J.N.Jansonius

Key ref:

V.N.Malashkevich et al. (1993). Crystal structures of true enzymatic reaction intermediates: aspartate and glutamate ketimines in aspartate aminotransferase. Biochemistry, 32, 13451-13462. PubMed id: 7903048 DOI: 10.1021/bi00212a010

Date:

10-Sep-93 Release date: 31-Jan-94

Protein chain

P00508  (AATM_CHICK) -  Aspartate aminotransferase, mitochondrial from Gallus gallus

Seq: 423 a.a.

Struc: 401 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 1: E.C.2.6.1.1 - aspartate transaminase.
• Reaction: L-aspartate + 2-oxoglutarate = oxaloacetate + L-glutamate
• Cofactor: Pyridoxal 5'-phosphate

Pyridoxal 5'-phosphate (Bound ligand (Het Group name = KET) matches with 60.00% similarity)

• Enzyme class 2: E.C.2.6.1.7 - kynurenine--oxoglutarate transaminase.

Pathway:

• Reaction: L-kynurenine + 2-oxoglutarate = kynurenate + L-glutamate + H2O
• Cofactor: Pyridoxal 5'-phosphate

Pyridoxal 5'-phosphate (Bound ligand (Het Group name = KET) matches with 60.00% similarity)

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1021/bi00212a010

Biochemistry 32:13451-13462 (1993)

PubMed id: 7903048

Crystal structures of true enzymatic reaction intermediates: aspartate and glutamate ketimines in aspartate aminotransferase.

V.N.Malashkevich, M.D.Toney, J.N.Jansonius.

ABSTRACT

The crystal structures of the stable, closed complexes of chicken mitochondrial aspartate aminotransferase with the natural substrates L-aspartate and L-glutamate have been solved and refined at 2.4- and 2.3-A resolution, respectively. In both cases, clear electron density at the substrate-coenzyme binding site unequivocally indicates the presence of a covalent intermediate. The crystallographically identical environments of the two subunits of the alpha 2 dimer allow a simple, direct correlation of the coenzyme absorption spectra of the crystalline enzyme with the diffraction results. Deconvolution of the spectra of the crystalline complexes using lognormal curves indicates that the ketimine intermediates constitute 76% and 83% of the total enzyme populations with L-aspartate and L-glutamate, respectively. The electron density maps accommodate the ketimine structures best in agreement with the independent spectral data. Crystalline enzyme has a much higher affinity for keto acid substrates compared to enzyme in solution. The increased affinity is interpreted in terms of a perturbation of the open/closed conformational equilibrium by the crystal lattice, with the closed form having greater affinity for substrate. The crystal lattice contacts provide energy required for domain closure normally supplied by the excess binding energy of the substrate. In solution, enzyme saturated with amino/keto acid substrate pairs has a greater total fraction of intermediates in the aldehyde oxidation state compared to crystalline enzyme. Assuming the only difference between the solution and crystalline enzymes is in conformational freedom, this difference suggests that one or more substantially populated, aldehydic intermediates in solution exist in the open conformation. Quantitative analyses of the spectra indicate that the value of the equilibrium constant for the open-closed conformational transition of the liganded, aldehydic enzyme in solution is near 1. The C4' pro-S proton in the ketimine models is oriented nearly perpendicularly to the plane of the pyridine ring, suggesting that the enzyme facilitates its removal by maximizing sigma-pi orbital overlap. The absence of a localized water molecule near Lys258 dictates that ketimine hydrolysis occurs via a transiently bound water molecule or from an alternative, possibly more open, structure in which water is appropriately bound. A prominent mechanistic role for flexibility of the Lys258 side chain is suggested by the absence of hydrogen bonds to the amino group in the aspartate structure and the relatively high temperature factors for these atoms in both structures.