title: Pressure and propylene carbonate denaturation of native and "aged" phosphorylated cholinesterase.
abstract: Organophosphate-inhibited cholinesterases may become progressively refractory to reactivation by nucleophilic compounds due to the dealkylation of an alkoxy group from the covalently bound phosphonate ester. This process is termed "aging". It has been found that "aged" cholinesterases are more resistant to protein unfolding than the non-inhibited ones. The pressure-induced denaturation of the native (non-inhibited) and "aged" tetrameric form of human plasma butyrylcholinesterase was investigated in the presence and absence of a denaturing agent (propylene carbonate). This study was undertaken to determine whether the stability of aged butyrylcholinesterase varies with the structure of the alkyl/aryl (R2) group remaining attached to the phosphorus atom of the organophosphoryl moiety. "Aged" organophosphoryl-cholinesterase conjugates were formed by reacting the enzyme with organophosphates: soman (trimethylpropylmethyl-phosphonofluoridate), sarin (isopropylmethyl-phosphonofluoridate), tabun (ethyl-N-dimethyl-phosphoramidocyanidate), DFP (diisopropyl phosphorofluoridate) and PBPDC (pyrenebutyl-phosphorodichloridate). The dual effects of hydrostatic pressure up to 3.5 kbar and propylene carbonate up to 1.2 M were investigated in 10 mM Tris.HCl (pH 7.0). Non-inhibited and aged enzymes were subjected to pressure/propylene carbonate for 12 hours at 20 degrees C. The perturbing effects of this treatment upon cholinesterase structure were analyzed after pressure release by non-denaturing electrophoresis. Pressure and propylene carbonate induced progressive inactivation of the native enzyme. The loss in activity was correlated with irreversible denaturation of the tetramer and its subsequent aggregation. Similarly, pressure and propylene carbonate induced the formation of irreversibly denatured forms of aged butyrylcholinesterase. These denatured forms are partially unfolded enzyme conformations. The native enzyme was found to be more susceptible to denaturation than aged enzymes, with the exception of the PBPDC-aged enzyme. Methyl phosphono adducts, i.e. soman or sarin-aged conjugates were found to be the most stable aged species. Phenomenological analysis of the pressure/propylene carbonate denaturation maps at half-way of the denaturation process indicated that denaturation is a multistep process. The lowest stability of tabun-aged and DFP-aged conjugates suggested that the size, the orientation and the hydrophobicity of the remaining alkyl/aryl chain (R2) of the organophosphoryl moiety play a role in determining the overall stability of aged enzymes. Molecular modelling of aged adducts shed light on steric constraints exerted by the R2 chain on the salt bridge formed between the negatively charged P-O- of the dealkylated organophosphoryl moiety and protonated His438 N epsilon.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Centre de Recherches, Service de Santé des Armées, La Tronche, France.
authors.forename: P
authors.initials: P
authors.lastname: Masson
authors.forename: P
authors.initials: P
authors.lastname: Gouet
authors.forename: C
authors.initials: C
authors.lastname: Clery
mesh_terms: D002091:Butyrylcholinesterase / Q000737:chemistry* / Q000378:metabolism; D002254:Carbonates / Q000494:pharmacology*; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D003196:Computer Graphics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D008958:Models, Molecular; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D011312:Pressure; D011407:Propane / Q000031:analogs & derivatives; D011487:Protein Conformation; D011489:Protein Denaturation
chemical_list: D002254:Carbonates; D002800:Cholinesterase Inhibitors; D009943:Organophosphorus Compounds; C045990:propylene carbonate; D002091:Butyrylcholinesterase; D011407:Propane