Open AccessNeutron structure and mechanistic studies of diisopropyl fluorophosphatase (DFPase)
Author(s) -
Chen Julian C.H.,
Mustyakimov Marat,
Schoenborn Benno P.,
Langan Paul,
Blum MarcMichael
Publication year - 2010
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
ISSN - 1399-0047
DOI - 10.1107/s0907444910034013
Subject(s) - chemistry , nucleophile , deprotonation , active site , protonation , nerve agent , stereochemistry , molecule , combinatorial chemistry , solvent , acetylcholinesterase , organic chemistry , enzyme , catalysis , ion
Diisopropyl fluorophosphatase (DFPase) is a calcium‐dependent phosphotriesterase that acts on a variety of highly toxic organophosphorus compounds that act as inhibitors of acetylcholinesterase. The mechanism of DFPase has been probed using a variety of methods, including isotopic labelling, which demonstrated the presence of a phosphoenzyme intermediate in the reaction mechanism. In order to further elucidate the mechanism of DFPase and to ascertain the protonation states of the residues and solvent molecules in the active site, the neutron structure of DFPase was solved at 2.2 Å resolution. The proposed nucleophile Asp229 is deprotonated, while the active‐site solvent molecule W33 was identified as water and not hydroxide. These data support a mechanism involving direct nucleophilic attack by Asp229 on the substrate and rule out a mechanism involving metal‐assisted water activation. These data also allowed for the re‐engineering of DFPase through rational design to bind and productively orient the more toxic S P stereoisomers of the nerve agents sarin and cyclosarin, creating a modified enzyme with enhanced overall activity and significantly increased detoxification properties.