The rate of thermal inactivation of <i>Torpedo</i> acetylcholinesterase is not reduced in the C231 S mutant

Wilson, Erica J.; Massoulié, Jean; Bon, Suzanne; Rosenberry, Terrone L.

doi:10.1016/0014-5793(95)01504-3

Cited by 13 publications

(15 citation statements)

References 21 publications

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“…A smaller decrease, to 135 kcal/mol, was observed for the W279A mutant, paralleling its more limited sensitivity to DTP. that the C231S mutant is slightly less stable than the WT and that the C231S/L282S double mutant is slightly less stable than L282S, which is in agreement with the observations of Wilson et al (1996). Vertebrate AChE displays a high degree of conservation, with Torpedo AChE possessing ca.…”

Section: Resultssupporting

confidence: 90%

X-Ray Crystallographic Studies on Acetylcholinesterase and Related Enzymes

Sussman¹,

Silman²

2003

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REPORT DOCUMENTATION PAGEForm ifl burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining ed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for lul&Vn to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and TITLE AND SUBTITLE X-Ray Crystallographic Studies on Acetylcholinesterase and Related Enzymes AUTHOR(S)Joel L. Sussman, Ph.D. I. Silman, Ph.D. FUNDING NUMBERS DAMD17-97-2-7022 PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)Weizmann Institute of Science Rehovot 76100 Israel E-MAIL: j oel.sussman@weizmann. ac.il Where copyrighted material is quoted, permission has been obtained to use such material. PERFORMING ORGANIZATION REPORT NUMBER SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES)UWhere material from documents designated for limited distribution is quoted, permission has been obtained to use the material.Citations of commercial organizations and trade names in this report do not constitute an official Department of Army endorsement or approval of the products or services of these organizations.N/A In conducting research using animals, the investigator(s) adhered to the "Guide for the Care and Use of Laboratory Animals," prepared by the Committee on Care and use of Laboratory Animals of the Institute of Laboratory Resources, national Research Council (NIH Publication No. 86-23, Revised 1985). N/A For the protection of human subjects, the investigator(s) adhered to policies of applicable Federal Law 45 CFR 46. N/A In conducting research utilizing recombinant DNA technology, the investigator(s) adhered to current guidelines promulgated by the National Institutes of Health. N/A Zh the conduct of research utilizing recombinant DNA, the investigator(s) adhered to the NIH Guidelines for Research Involving Recombinant DNA Molecules. N/A In the conduct of research involving hazardous organisms, the investigator(s) adhered to the CDC-NIH Guide for Biosafety in Microbiological and Biomedieal Laboratories.l->r*iA. J J*-»

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Section: Resultssupporting

confidence: 90%

X-Ray Crystallographic Studies on Acetylcholinesterase and Related Enzymes

Sussman¹,

Silman²

2003

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“…X-ray crystallography showed that apart from space adequate to accommodate five or six water molecules, a single DECA molecule defines and fills the volume of the gorge [29]. Inactivation of wt-Torpedo californica AChE was 4 times faster in phosphate buffer than in Tris buffer, a fact consistent with AChE stabilization by cationic Tris bound to the active site [31]. Catalytic antibodies can be generated by immunization with haptens [32] and in antiidiotypic antibodies against anti-enzyme antibodies [33,34].…”

Section: Incidence Of the Nature And Composition Of Analytesmentioning

confidence: 78%

Detection of unwanted protein-bound ligands by capillary zone electrophoresis: The case of hidden ligands that stabilize cholinesterase conformation

2002

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Detection, identification and characterization of compounds present in purified proteins and biopharmaceuticals are of central interest. As well as chemical remedies, proteins of pharmacological interest have to exhibit their nakedness to become therapeutic drugs. Cholinesterases (ChE) are enzymes of major importance for detoxification of poisonous esters. Likewise, ChE are characterized by the high catalytic efficiency of an active site positioned at the bottom of a deep gorge. The gorge can be partially or fully occupied by ligands, i.e., substrates and inhibitors that are currently used in affinity chromatography purification steps. Accordingly, a suitable method allowing to analyse the presence of unwanted ligands and its influence on the functional conformation and stability of these enzymes was essential. We have developed CZE approaches for that purpose. The factors causing discrepancies between data for thermal unfolding of ChE by electrophoretic and by calorimetric methods were investigated. The presence of unwanted hidden ligands bound to purified enzymes was first demonstrated. The incidence of these ligands was discussed. Altogether, our results raised several questions concerning the real conformation of the native state of enzymes. Finally, CZE was proved to be a pertinent tool to validate the conformity of purified enzymes to a status of biopharmaceutical.

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“…The kinetics for ChE2 are confounded by the presence of the two cysteines. Wilson et al (1996) studied the inactivation of wild type and C231S Torpedo AChE by DTNB. Some of their results are different from ours.…”

Section: Discussionmentioning

confidence: 99%

“…The AChE from T. californica can be inactivated by sulfhydryl reagents, apparently by the reagents reacting stoichiometrically with a free cysteine residue, Cys231, to form a mixed disulfide (Steinberg et al 1990;Dolginova et al 1992;Wilson et al 1996;Morel et al 1999). Notably, Cys231 is in the catalytic gorge of AChE, approximately 8 Å from the active site serine (Morel et al 1999).…”

Section: Introductionmentioning

confidence: 96%

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

et al. 2006

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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.

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The rate of thermal inactivation of Torpedo acetylcholinesterase is not reduced in the C231 S mutant

Cited by 13 publications

References 21 publications

X-Ray Crystallographic Studies on Acetylcholinesterase and Related Enzymes

X-Ray Crystallographic Studies on Acetylcholinesterase and Related Enzymes

Detection of unwanted protein-bound ligands by capillary zone electrophoresis: The case of hidden ligands that stabilize cholinesterase conformation

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

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