1QIR

ASPARTATE AMINOTRANSFERASE FROM ESCHERICHIA COLI, C191Y MUTATION, WITH BOUND MALEATE

1QIR の概要

• エントリーDOI: 10.2210/pdb1qir/pdb
• 関連するPDBエントリー: 1QIS 1QIT
• 分子名称: ASPARTATE AMINOTRANSFERASE, PYRIDOXAL-5'-PHOSPHATE, MALEIC ACID, ... (4 entities in total)
• 機能のキーワード: aminotransferase, transferase(aminotransferase), pyridoxal phosphate, maleate
• 由来する生物種: ESCHERICHIA COLI
• 細胞内の位置: Cytoplasm: P00509
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 44042.46

構造登録者

Jeffery, C.J.,Gloss, L.M.,Petsko, G.A.,Ringe, D. (登録日: 1999-06-15, 公開日: 2000-06-05, 最終更新日: 2023-12-13)

主引用文献

Jeffery, C.J.,Gloss, L.M.,Petsko, G.A.,Ringe, D.
The Role of Residues Outside the Active Site in Catalysis: Structural Basis for Function of C191 Mutants of E. Coli Aspartate Aminotransferase
Protein Eng., 13:105-, 2000

PubMed 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.

PubMed: 10708649
DOI: 10.1093/PROTEIN/13.2.105

実験手法

X-RAY DIFFRACTION (2.2 Å)