Back to Search
Start Over
The Architecture of Human Acetylcholinesterase Active Center Probed by Interactions with Selected Organophosphate Inhibitors
- Source :
- Journal of Biological Chemistry. 271:11953-11962
- Publication Year :
- 1996
- Publisher :
- Elsevier BV, 1996.
-
Abstract
- The role of the functional architecture of human acetylcholinesterase (HuAChE) active center in facilitating reactions with organophosphorus inhibitors was examined by a combination of site-directed mutagenesis and kinetic studies of phosphorylation with organophosphates differing in size of their alkoxy substituents and in the nature of the leaving group. Replacements of residues Phe-295 and Phe-297, constituting the HuAChE acyl pocket, increase up to 80-fold the reactivity of the enzymes toward diisopropyl phosphorofluoridate, diethyl phosphorofluoridate, and p-nitrophenyl diethyl phosphate (paraoxon), indicating the role of this subsite in accommodating the phosphate alkoxy substituent. On the other hand, a decrease of up to 160-fold in reactivity was observed for enzymes carrying replacements of residues Tyr-133, Glu-202, and Glu-450, which are constituents of the hydrogen bond network in the HuAChE active center, which maintains its unique functional architecture. Replacement of residues Trp-86, Tyr-337, and Phe-338 in the alkoxy pocket affected reactivity toward diisopropyl phosphorofluoridate and paraoxon, but to a lesser extent that toward diethyl phosphorofluoridate, indicating that both the alkoxy substituent and the p-nitrophenoxy leaving group interact with this subsite. In all cases the effects on reactivity toward organophosphates, demonstrated in up to 10,000-fold differences in the values of bimolecular rate constants, were mainly a result of altered affinity of the HuAChE mutants, while the apparent first order rate constants of phosphorylation varied within a narrow range. This finding indicates that the main role of the functional architecture of HuAChE active center in phosphorylation is to facilitate the formation of enzyme-inhibitor Michaelis complexes and that this affinity, rather than the nucleophilic activity of the enzyme catalytic machinery, is a major determinant of HuAChE reactivity toward organophosphates.
- Subjects :
- Stereochemistry
Substituent
Biochemistry
Active center
Structure-Activity Relationship
chemistry.chemical_compound
Organophosphorus Compounds
medicine
Humans
Reactivity (chemistry)
Phosphorylation
Molecular Biology
Binding Sites
Paraoxon
Organophosphate
Leaving group
Hydrogen Bonding
Cell Biology
Acetylcholinesterase
Kinetics
chemistry
Mutagenesis, Site-Directed
Alkoxy group
Cholinesterase Inhibitors
medicine.drug
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 271
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....fd1141061e8438efd3a0b8cf43d041f3