title: The pH dependence of dealkylation in soman-inhibited cholinesterases and their mutants: further evidence for a push-pull mechanism.
abstract: Bimolecular rate constants for the inactivation of recombinant (r) human (Hu) butyrylcholinesterase (BChE) with P(S)C(S)- and P(S)C(R)-2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) are (92 +/- 7) x 10(6) M-1 min-1 and (13.7 +/- 0.8) x 10(6) M-1 min-1 at pH 7.4, mu = 0.1 M and 25 degreesC. Mutations of E197(199) to D or Q and W82(84) to A result in reductions in the rate constants for inactivation with P(S)C(S)-soman 4.3-, 11.8-, and 263-fold and with P(S)C(R)-soman by 6.5-, 47.3-, and 685-fold, respectively. The pH dependence of dealkylation (aging) in r mouse (Mo) acetylcholinesterase (AChE) and rHu BChE and their mutants inactivated with P(S)C(S)- and P(S)C(R)-soman was compared. Best-fit parameters for the asymmetric bell curves for the adducts of wild-type Mo AChE are pK1 = pK2 = 4.0-4.9 and pK3 = 5.2-6.6. These pKs are consistent with the involvement of two carboxylic acids, possibly E202(199) and either E334(327) or E450(443), and H447(440)H+ in the dealkylation of AChE. E202Q MoAChE inactivated with the soman diastereomers yielded pK3 = 5.5-5.8. Nearly symmetric pH curves for soman-inhibited wild-type and E197D Hu BChE gave pK2 = 3.7-4.6 and pK3 = 7.3-8.0, but much lower, pK3 approximately 5, for the corresponding adduct of the E197Q mutant. Dealkylation in soman-inhibited BChE is consistent with the participation of one carboxylic acid side chain and H438(440)H+. Maximal rate constants for dealkylation (kmax) are 1-6 min-1 for AChE and 2 min-1 for BChE at 25 degreesC. The W82 to A mutation in BChE results in the largest reduction, 2500-6000-fold, in the rate constant for dealkylation. The reduction in the rate constants for dealkylation in the E197 mutants is highly pH dependent. The solvent isotope effects at the pH maxima are 1.3-1.4, indicating unlikely preprotonation or proton in "flight" at the enzymic transition states. The new results support the push-pull mechanism of dealkylation in soman-inhibited cholinesterases proposed previously.
authors.affiliation: Division of Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307, USA.
authors.forename: A
authors.initials: A
authors.lastname: Saxena
authors.forename: C
authors.initials: C
authors.lastname: Viragh
authors.forename: D S
authors.initials: DS
authors.lastname: Frazier
authors.forename: I M
authors.initials: IM
authors.lastname: Kovach
authors.forename: D M
authors.initials: DM
authors.lastname: Maxwell
authors.forename: O
authors.initials: O
authors.lastname: Lockridge
authors.forename: B P
authors.initials: BP
authors.lastname: Doctor
mesh_terms: D000110:Acetylcholinesterase / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002091:Butyrylcholinesterase / Q000235:genetics* / Q000378:metabolism*; D002800:Cholinesterase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D003640:Dealkylation; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012999:Soman / Q000737:chemistry / Q000494:pharmacology*; D013237:Stereoisomerism; D013997:Time Factors; D014364:Tryptophan / Q000235:genetics
chemical_list: D002800:Cholinesterase Inhibitors; D011994:Recombinant Proteins; D018698:Glutamic Acid; D014364:Tryptophan; D012999:Soman; D000110:Acetylcholinesterase; D002091:Butyrylcholinesterase