Accession Number : ADA319311

Title :   Production and Enzyme Engineering of Human Acetylcholinesterase and Its Mutant Derivatives.

Descriptive Note : Final rept. 15 Jun 93-31 Mar 96,

Corporate Author : ISRAEL INST FOR BIOLOGICAL RESEARCH NESS ZIONA

Personal Author(s) : Shafferman, Avigdor

PDF Url : ADA319311

Report Date : MAY 1996

Pagination or Media Count : 277

Abstract : Studies toward the design of pharmacologically useful bio-scavengers against organophosphate poisoning, were pursued in two main directions: (a) structure-reactivity characteristics of human acerylcholinesterase (HuAChE) derivatives and of the corresponding phosphyl conjugates; (b) molecular surface properties of HuAChE derivatives affecting biosynthesis, stability and clearance. Combination of recombinant DNA technology, kinetic studies and molecular modeling was employed to identify some of the residues involved In direct interactions with organophosphates and phosphonates and in the subsequent aging processes. Our findings allow to define the structural determinants in the active center facilitating the bio-scavenging and conferring resistance to aging. In addition, we identified residues at the peripheral anionic. site (PAS), participating in modulation of HuAChE reactivity, and defined a possible structural link for transmission of the allosteric signal to the active center. Such mechanism probably involves the conformational mobility of the surface loop (Cys69-Cys96) containing specific elements of the PAS and the active center. Alterations of the molecular surface properties of HuAChE including replacements of up to seven acidic residues, failed to alter significantly the catalytic properties of the mutants relative to the wild type HuAChE. Manipulation of the level of glycosylation sites by mutagenesis affected biosynthesis, secretion, thermal stability and the rates of clearance. It was demonstrated that a control over the number of vacant sialic acid attachment sites may improve the enzyme residence time in the bloodstream.

Descriptors :   *DEOXYRIBONUCLEIC ACIDS, *GENETIC ENGINEERING, *ACETYLCHOLINESTERASE, *ORGANOPHOSPHATES, PRODUCTION, AGING(MATERIALS), MODELS, HUMANS, INTERACTIONS, MOLECULES, RESISTANCE, ENZYMES, SITES, REACTIVITIES, THERMAL STABILITY, MUTATIONS, SURFACES, ENGINEERING, SURFACE PROPERTIES, CATALYSIS, POISONING, ATTACHMENT, KINETICS, MODULATION, LOOPS, PHOSPHONATES, WILDLIFE, MOLECULAR PROPERTIES, RESIDUES, BIOSYNTHESIS, PHARMACOLOGY, ACIDS, SIALIC ACIDS.

Subject Categories : Biochemistry
      Genetic Engineering and Molecular Biology
      Pharmacology

Distribution Statement : APPROVED FOR PUBLIC RELEASE