Determinants of substrate specificity of a second non-neuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis

We recently reported on a non-neuronal secreted acetylcholinesterase (AChE B) from the nematode parasite Nippostrongylus brasiliensis. Here we describ e the primary structure and enzymatic properties of a second secreted varia nt, termed AChE C after the designation of native AChE isoforms from this p arasite. As for the former enzyme, AChE C is truncated at the carboxyl term inus in comparison with the Torpedo AChE, and three of the 14 aromatic resi dues that line the active site gorge are substituted by nonaromatic residue s, corresponding to Tyr70 (Ser), Trp279 (Asn) and Phe288 (Met). A recombinant form of AChE C was highly expressed by Pichia pastoris. The e nzyme was monomeric and hydrophilic, and displayed a marked preference for acetylthiocholine as substrate. A double mutation (W302F/W345F, correspondi ng to positions 290 and 331 in Torpedo) rendered the enzyme 10-fold less se nsitive to excess substrate inhibition and two times less susceptible to th e bis quaternary inhibitor BW284C51, but did not radically affect substrate specificity or sensitivity to the 'peripheral site' inhibitor propidium io dide. In contrast, a triple mutant (M300G/W302F/W345F) efficiently hydrolys ed propionylthiocholine and butyrylthiocholine in addition to acetylthiocho line, while remaining insensitive to the butyrylcholinesterase-specific inh ibitor iso-OMPA and displaying a similar profile of excess substrate inhibi tion as the double mutant. These data highlight a conserved pattern of acti ve site architecture for nematode secreted AChEs characterized to date, and provide an explanation for the substrate specificity that might otherwise appear inconsistent with the primary structure in comparison to other inver tebrate AChEs.