4EKQ

T4 Lysozyme L99A/M102H with 4-Nitrophenol Bound

4EKQ の概要

• エントリーDOI: 10.2210/pdb4ekq/pdb
• 関連するPDBエントリー: 4E97 4EKP 4EKR 4EKS
• 分子名称: Lysozyme, BETA-MERCAPTOETHANOL, P-NITROPHENOL, ... (7 entities in total)
• 機能のキーワード: hydrolase, alkylation of cys97
• 由来する生物種: Enterobacteria phage T4
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 44076.06

構造登録者

Merski, M.,Shoichet, B.K. (登録日: 2012-04-09, 公開日: 2012-09-05, 最終更新日: 2023-09-13)

主引用文献

Merski, M.,Shoichet, B.K.
Engineering a model protein cavity to catalyze the Kemp elimination.
Proc.Natl.Acad.Sci.USA, 109:16179-16183, 2012

PubMed Abstract: Synthetic cavitands and protein cavities have been widely studied as models for ligand recognition. Here we investigate the Met102 → His substitution in the artificial L99A cavity in T4 lysozyme as a Kemp eliminase. The resulting enzyme had k(cat)/K(M) = 0.43 M(-1) s(-1) and a (k(cat)/K(M))/k(uncat) = 10(7) at pH 5.0. The crystal structure of this enzyme was determined at 1.30 Å, as were the structures of four complexes of substrate and product analogs. The absence of ordered waters or hydrogen bonding interactions, and the presence of a common catalytic base (His102) in an otherwise hydrophobic, buried cavity, facilitated detailed analysis of the reaction mechanism and its optimization. Subsequent substitutions increased eliminase activity by an additional four-fold. As activity-enhancing substitutions were engineered into the cavity, protein stability decreased, consistent with the stability-function trade-off hypothesis. This and related model cavities may provide templates for studying protein design principles in radically simplified environments.

PubMed: 22988064
DOI: 10.1073/pnas.1208076109

実験手法

X-RAY DIFFRACTION (1.54 Å)