PDBsum entry 1b4x

Transferase

PDB id: 1b4x

Contents

Protein chain

396 a.a. *

Ligands

PLP

MAE

Waters ×26

* Residue conservation analysis

PDB id:

1b4x

Name:

Transferase

Title:

Aspartate aminotransferase from e. Coli, c191s mutation, with bound maleate

Structure:

Aspartate aminotransferase. Chain: a. Fragment: complete subunit. Engineered: yes. Mutation: yes. Other_details: pyridoxal phosphate cofactor covalently bound to lys 258 via schiff base linkage

Source:

Escherichia coli. Organism_taxid: 562. Cellular_location: cytoplasm. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.45Å R-factor: 0.207

Authors:

C.J.Jeffery,L.M.Gloss,G.A.Petsko,D.Ringe

Key ref:

C.J.Jeffery et al. (2000). The role of residues outside the active site: structural basis for function of C191 mutants of Escherichia coli aspartate aminotransferase. Protein Eng, 13, 105-112. PubMed id: 10708649

Date:

30-Dec-98 Release date: 27-Oct-00

Protein chain

P00509  (AAT_ECOLI) -  Aspartate aminotransferase from Escherichia coli (strain K12)

Seq: 396 a.a.

Struc: 396 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.2.6.1.1 - aspartate transaminase.
• Reaction: L-aspartate + 2-oxoglutarate = oxaloacetate + L-glutamate

L-aspartate + 2-oxoglutarate (Bound ligand (Het Group name = MAE) matches with 88.89% similarity) = oxaloacetate + L-glutamate

• Cofactor: Pyridoxal 5'-phosphate

Pyridoxal 5'-phosphate (Bound ligand (Het Group name = PLP) matches with 93.75% similarity)

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

reference

Protein Eng 13:105-112 (2000)

PubMed id: 10708649

The role of residues outside the active site: structural basis for function of C191 mutants of Escherichia coli aspartate aminotransferase.

C.J.Jeffery, L.M.Gloss, G.A.Petsko, D.Ringe.

ABSTRACT

In previous kinetic studies of Escherichia coli aspartate aminotransferase, it was determined that some substitutions of conserved cysteine 191, which is located outside of the active site, altered the kinetic parameters of the enzyme (Gloss,L.M., Spencer,D. E. and Kirsch,J.F., 1996, Protein Struct. Funct. Genet., 24, 195-208). The mutations resulted in an alkaline shift of 0.6-0.8 pH units for the pK(a) of the internal aldimine between the PLP cofactor and Lys258. The change in the pK(a) affected the pH dependence of the k(cat)/K(m) (aspartate) values for the mutant enzymes. To help to understand these observations, crystal structures of five mutant forms of E.coli aspartate aminotransferase (the maleate complexes of C191S, C191F, C191Y and C191W, and C191S without maleate) were determined at about 2 A resolution in the presence of the pyridoxal phosphate cofactor. The overall three-dimensional fold of each mutant enzyme is the same as that of the wild-type protein, but there is a rotation of the mutated side chain around its C(alpha)-C(beta) bond. This side chain rotation results in a change in the pattern of hydrogen bonding connecting the mutant residue and the protonated Schiff base of the cofactor, which could account for the altered pK(a) of the Schiff base imine nitrogen that was reported previously. These results demonstrate how residues outside the active site can be important in helping determine the subtleties of the active site amino acid geometries and interactions and how mutations outside the active site can have effects on catalysis. In addition, these results help explain the surprising result previously reported that, for some mutant proteins, replacement of a buried cysteine with an aromatic side chain did not destabilize the protein fold. Instead, rotation around the C(alpha)-C(beta) bond allowed each large aromatic side chain to become buried in a nearby pocket without large changes in the enzyme's backbone geometry.