P. Masson et al., IMPORTANCE OF ASPARTATE-70 IN ORGANOPHOSPHATE INHIBITION, OXIME REACTIVATION AND AGING OF HUMAN BUTYRYLCHOLINESTERASE, Biochemical journal, 325, 1997, pp. 53-61
Asp-70 is the defining amino acid in the peripheral anionic site of hu
man butyrylcholinesterase (BuChE), whereas acetylcholinesterase has se
veral additional amino acids, the most important one being Trp-277 (Tr
p-279 in Torpedo AChE). We studied mutants D70G, D70K and A277W to eva
luate the role of Asp-70 and Trp-277 in reactions with organophosphate
s. We found that Asp-70 was important for binding positively charged e
chothiophate, but not neutral paraoxon and iso-OMPA. Asp-70 was also i
mportant for binding of positively charged pralidoxime (2-PAM) and for
activation of re-activation by excess 2-PAM. Excess 2-PAM had an effe
ct similar to Substrate activation, suggesting the binding of 2 mol of
2-PAM to wild-type but not to the D70G mutant. A. surprising result w
as that Asp-70 was important for irreversible aging, the D70G mutant h
aving a 3- and 8-fold lower rate of aging for paraoxon-inhibited and d
i-isopropyl fluorophosphate-inhibited BuChE. Mutants of Asp-70 had the
same rate constants for phosphorylation and re-activation by 2-PAM as
wild-type. The A277W mutant behaved like wild-type in all assays. Our
results predict that people with the atypical (D70G) variant of BuChE
will be more sensitive to the toxic effects of echothiophate, but wil
l be equally sensitive to paraoxon and di-isopropyl fluorophosphate. P
eople with the D70G mutation will be resistant to re-activation of the
ir inhibited BuChE by 2-PAM, but this will be offset by the lower rate
of irreversible aging of inhibited BuChE, allowing some regeneration
by spontaneous hydrolysis.