1AIA

STRUCTURAL BASIS FOR THE CATALYTIC ACTIVITY OF ASPARTATE AMINOTRANSFERASE K258H LACKING THE PYRIDOXAL-5'-PHOSPHATE BINDING LYSINE RESIDUE

1AIA の概要

• エントリーDOI: 10.2210/pdb1aia/pdb
• 分子名称: ASPARTATE AMINOTRANSFERASE, 4'-DEOXY-4'-AMINOPYRIDOXAL-5'-PHOSPHATE (2 entities in total)
• 機能のキーワード: transferase(aminotransferase)
• 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: 2
• 化学式量合計: 87752.71

構造登録者

Jaeger, J.,Jansonius, J.N. (登録日: 1994-05-10, 公開日: 1994-10-15, 最終更新日: 2024-02-07)

主引用文献

Malashkevich, V.N.,Jager, J.,Ziak, M.,Sauder, U.,Gehring, H.,Christen, P.,Jansonius, J.N.
Structural basis for the catalytic activity of aspartate aminotransferase K258H lacking the pyridoxal 5'-phosphate-binding lysine residue.
Biochemistry, 34:405-414, 1995

PubMed Abstract: Chicken mitochondrial and Escherichia coli aspartate aminotransferases K258H, in which the active site lysine residue has been exchanged for a histidine residue, retain partial catalytic competence [Ziak et al. (1993) Eur. J. Biochem. 211, 475-484]. Mutant PLP and PMP holoenzymes and the complexes of the latter (E. coli enzyme) with sulfate and 2-oxoglutarate, as well as complexes of the mitochondrial apoenzyme with N-(5'-phosphopyridoxyl)-L-aspartate or N-(5'-phosphopyridoxyl)-L-glutamate, were crystallized and analyzed by means of X-ray crystallography in order to examine how the side chain of histidine 258 can substitute as a general acid/base catalyst of the aldimine-ketimine tautomerization in enzymic transamination. The structures have been solved and refined at resolutions between 2.1 and 2.8 A. Both the closed and the open conformations, identical to those of the wild-type enzyme, were observed, indicating that the mutant enzymes of both species exhibit the same conformational flexibility as the wild-type enzymes, although in AspAT K258H the equilibrium is somewhat shifted toward the open conformation. The replacement of the active site K258 by a histidine residue resulted only in local structural adaptations necessary to accommodate the imidazole ring. The catalytic competence of the mutant enzyme, which in the forward half-reaction is 0.1% of that of the wild-type enzyme, suggests that the imidazole group is involved in the aldimine-ketimine tautomerization. However, the imidazole ring of H258 is too far away from C alpha and C4' of the coenzyme-substrate adduct for direct proton transfer, suggesting that the 1,3-prototropic shift is mediated by a water molecule. Although there is enough space for a water molecule in this area, it has not been detected. Dynamic fluctuations of the protein matrix might transiently open a channel, giving a water molecule fleeting access to the active site.

PubMed: 7819232
DOI: 10.1021/bi00002a004

実験手法

X-RAY DIFFRACTION (2.2 Å)