1USB

Rational design of a novel enzyme - efficient thioester hydrolysis enabled by the incorporation of a single His residue into human glutathione transferase A1-1

1USB の概要

• エントリーDOI: 10.2210/pdb1usb/pdb
• 関連するPDBエントリー: 1GSD 1GSE 1GSF 1GUH 1K3L 1K3O 1K3Y 1LBK
• 分子名称: GLUTATHIONE S-TRANSFERASE A1, GLUTATHIONE, CHLORIDE ION, ... (5 entities in total)
• 機能のキーワード: transferase, glutathione
• 由来する生物種: HOMO SAPIENS (HUMAN)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 52608.36

構造登録者

Jakobsson, E.,Kleywegt, G.J. (登録日: 2003-11-20, 公開日: 2004-09-01, 最終更新日: 2023-12-13)

主引用文献

Hederos, S.,Broo, K.S.,Jakobsson, E.,Kleywegt, G.J.,Mannervik, B.,Baltzer, L.
Incorporation of a Single His Residue by Rational Design Enables Thiol-Ester Hydrolysis by Human Glutathione Transferase A1-1
Proc.Natl.Acad.Sci.USA, 101:13163-, 2004

PubMed Abstract: A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.

PubMed: 15333749
DOI: 10.1073/PNAS.0403045101

実験手法

X-RAY DIFFRACTION (2.07 Å)