A tryptophan in the bottleneck of the catalytic gorge of an invertebrate acetylcholinesterase confers relative resistance to carbamate and organophosphate inhibitors.
Amphioxus, an invertebrate chordate, has two acetylcholinesterases (AChEs): cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2). ChE1 is up to 329-fold more resistant to a variety of carbamate and organophosphate inhibitors, including a number of insecticides, when compared with ChE2. One difference between the two enzymes is at the position homologous to Phe331 in Torpedo AChE. In Torpedo AChE, this residue is a component of the hydrophobic subsite and defines one side of the bottleneck in the catalytic gorge of the enzyme. In ChE1, the homologous residue is Trp353; in ChE2, it is Phe353. We used site-directed mutagenesis to investigate the proposal that the resistance of ChE1 to inhibition by carbamates and organophosphates was due to this difference, creating a ChE1 W353F mutant to widen the bottleneck. The mutation virtually abolishes the difference in sensitivity to the inhibitors. The ChE1 W353F mutant is only 2- to 3-fold more resistant than ChE2 to carbamates and is actually 2.5- to 10-fold more sensitive to inhibition by organophosphates. The differences in resistance are due to different affinities of the enzymes for the inhibitors, not different reactivities. Molecular modeling supports the proposal that the difference in inhibition is due to the width of the bottleneck of the gorge. Our results have implications for insecticide resistance in insects, in particular mosquitoes and aphids.
Patel
Raj
R
Division of Science and Mathematics, Birmingham-Southern College, Box 549022, Birmingham, AL 35254, USA.
Sanders
Rachael
R
Brown
Lance
L
Baker
Stephanie
S
Tsigelny
Igor
I
Pezzementi
Leo
L
D000110:Acetylcholinesterase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002219:Carbamates / Q000494:pharmacology*; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D002802:Cholinesterases / Q000235:genetics / Q000378:metabolism*; D002816:Chordata, Nonvertebrate / Q000201:enzymology*; D004789:Enzyme Activation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010755:Organophosphates / Q000494:pharmacology*; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics*
D002219:Carbamates; D002800:Cholinesterase Inhibitors; D010755:Organophosphates; D014364:Tryptophan; C116696:cholinesterase 1; C116697:cholinesterase 2; D000110:Acetylcholinesterase; D002802:Cholinesterases