Invertebrate Neuroscience

, Volume 6, Issue 2, pp 47–55 | Cite as

Inactivation of an invertebrate acetylcholinesterase by sulfhydryl reagents: the roles of two cysteines in the catalytic gorge of the enzyme

  • Leo Pezzementi
  • Melissa Rowland
  • Matthew Wolfe
  • Igor Tsigelny
Original Paper


We have used site-directed mutagenesis and molecular modeling to investigate the inactivation of an invertebrate acetylcholinesterase (AChE), ChE2 from amphioxus, by the sulfhydryl reagents 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM), creating various mutants, including C310A and C466A, and the double mutants C310A/C466A and C310A/F312I, to assess the relative roles of the two cysteines and a proposal that the increased rate of inactivation in the F312I mutant is due to increased access to Cys310. Our results suggest that both cysteines may be involved in inactivation by sulfhydryl reagents, but that the cysteine in the vicinity of the acyl pocket is more accessible. We speculate that the inactivation of aphid AChEs by sulfhydryl reagents is due to the presence of a cysteine homologous to Cys310. We also investigated the effects of various reversible cholinergic ligands, which bind to different subsites of the active site of the enzyme, on the rate of inactivation by DTNB of wild type ChE2 and ChE2 F312I. For the most part the inhibitors protect the enzymes from inactivation by DTNB. However, a notable exception is the peripheral site ligand propidium, which accelerates inactivation in the wild type ChE2, but retards inactivation in the F312I mutant. We propose that these opposing effects are the result of an altered allosteric signal transduction mechanism in the F312I mutant compared to the wild type ChE2.


Acetylcholinesterase Cysteine Sulfhydryl reagents Aphids Amphioxus 



This work was supported by Academic Research Award No. 1 R15 GM072510-01 from the National Institutes of Health to L.P. Additional support was provided by Birmingham-Southern College.


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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Leo Pezzementi
    • 1
  • Melissa Rowland
    • 1
  • Matthew Wolfe
    • 1
  • Igor Tsigelny
    • 2
  1. 1.Division of Science and MathematicsBirmingham-Southern CollegeBirminghamUSA
  2. 2.Department of Chemistry and Biochemistry, and San Diego Supercomputer CenterUniversity of California at San DiegoLa JollaUSA

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