1NJ9

Cocaine hydrolytic antibody 15A10

1NJ9 の概要

• エントリーDOI: 10.2210/pdb1nj9/pdb
• 分子名称: immunoglobulin variable chain, immunoglobulin heavy chain, SODIUM ION, ... (4 entities in total)
• 機能のキーワード: immunoglobulin, immune system
• 由来する生物種: Mus musculus (house mouse); 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 92176.27

構造登録者

Larsen, N.A.,de Prada, P.,Deng, S.X.,Zhu, X.,Landry, D.W.,Wilson, I.A. (登録日: 2002-12-30, 公開日: 2004-02-17, 最終更新日: 2024-10-30)

主引用文献

Larsen, N.A.,de Prada, P.,Deng, S.X.,Mittal, A.,Braskett, M.,Zhu, X.,Wilson, I.A.,Landry, D.W.
Crystallographic and biochemical analysis of cocaine-degrading antibody 15A10.
Biochemistry, 43:8067-8076, 2004

PubMed Abstract: Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.

PubMed: 15209502
DOI: 10.1021/bi049495l

実験手法

X-RAY DIFFRACTION (2.35 Å)