Kinetic, equilibrium, and spectroscopic studies on dealkylation ("aging") of alkyl organophosphonyl acetylcholinesterase. Electrostatic control of enzyme topography.
The mechanism of dealkylation ("aging") of branched-alkyl organophosphonyl conjugates of acetylcholinesterase and the consequence of this reaction on enzyme conformation were examined by employing kinetic, equilibrium, and spectroscopic techniques. Aging of cycloheptyl methylphosphono-acetylcholinesterase proceeded as a unimolecular reaction in which the enzyme became refractory to oxime reactivation and was accelerated with increases in temperature and decreases in pH and ionic strength of the medium. While aging occurred in a manner invariant with the nature of the salt in buffers containing Na+, K+, Rb+, Cs+, Cl-, CH3COO-, SO2-(4), and PO3-(4), the influence of ionic strength on aging was opposite to that predicted for a mechanism requiring charge separation during formation of the polar transition state. Examination of the equilibrium enzyme conformation with decidium, a fluorescent active center-selective ligand, revealed marked alterations in ligand association and a greater ionic strength dependence for binding after aging. The explanation for this behavior focuses on the high net negative surface charge of the enzyme and proposes that acetylcholinesterase topography is governed by the strength of electrostatic interactions between charged, contiguous, mobile protein regions within the subunit. As such, these studies reveal a reciprocal relationship between acetylcholinesterase topography, surface charge, and ionic strength of the medium.
D000110:Acetylcholinesterase / Q000378:metabolism*; D000478:Alkylation; D000818:Animals; D004491:Edrophonium / Q000494:pharmacology; D004557:Electric Organ / Q000201:enzymology; D007700:Kinetics; D008024:Ligands; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010617:Phenanthridines / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D014101:Torpedo
D008024:Ligands; D009943:Organophosphorus Compounds; D010617:Phenanthridines; D000644:Quaternary Ammonium Compounds; C049749:decidium; C009571:phenyltrimethylammonium; D004491:Edrophonium; D000110:Acetylcholinesterase