Unique push-pull mechanism of dealkylation in soman-inhibited cholinesterases.
The pH-dependence and solvent isotope effects of dealkylation in diastereomeric adducts of Electric eel (Ee) and fetal bovine serum (FBS) acetylcholinesterase (AChE) inactivated with P(-)C(+) and P(-)C(-) 2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) were studied at 4.0 +/- 0.2 degrees C. The rate constant versus pH profiles were fit to a bell-shaped curve for all adducts. Best fit parameters are pK1 4.4-4.6 and pK2 6.3-6.5 for Ee AChE and pK1 4.8-5. 0 and pK2 5.8 for FBS AChE. The pKs are consistent with catalytic participation of the Glu199 anion and HisH+440. Maximal rate constants (kmax) are 13-16 x 10(-3) s-1 for Ee AChE and 8 x 10(-3) s-1 for FBS AChE. The solvent isotope effects at the pH maxima are 1.1-1.3, indicating unlikely proton transfer at the enzymic transition states for the dealkylation reaction. Slopes of log rate constant versus pH plots are near 1 at 25.0 +/- 0.2 degrees C between pH 7.0 and 10.0. In stark contrast, the corresponding adducts of trypsin are very stable even at 37.0 +/- 0.2 degrees C. The rate constants for diastereomers of soman-inhibited trypsin at 37.0 +/- 0.2 degrees C are pH independent and approximately 10(4) times smaller than kmax for analogous adducts with AChE. Dealkylation in soman-inhibited AChEs is estimated to occur at >10(10) times faster than a plausible nonenzymic reaction. Up to 40% of the catalytic acceleration can be attributed to an electrostatic push, and an electrostatic pull provides much of the balance. The results of this work together with results of a product analysis by Michel et al. (1969) can be explained by an initial and rate-determining methyl migration from Cbeta to Calpha. This is driven by the high electron density of residues (Glu199 and Trp84) at a crowded active site and may be concerted with C-O bond breaking. The positive charge at the rate-determining transition state is distributed between Cbeta and His440. A tertiary carbocation may have a fleeting existence before it is trapped by water or neighboring electrons which is likely to be promoted by Glu199 as the proton acceptor.
Viragh
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U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5425, USA.
Akhmetshin
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Kovach
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Broomfield
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D000110:Acetylcholinesterase / Q000737:chemistry* / Q000378:metabolism*; D000478:Alkylation; D000818:Animals; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D004524:Eels; D004563:Electrochemistry; D004789:Enzyme Activation / Q000187:drug effects; D005312:Fetal Blood / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012997:Solvents; D012999:Soman / Q000494:pharmacology*; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism
D002800:Cholinesterase Inhibitors; D012997:Solvents; D012999:Soman; D000110:Acetylcholinesterase