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|Title:||Biochemical characterisation of the catalytic domain of neuropathy target esterase|
|Presented at:||University of Leicester|
|Abstract:||Neuropathy target esterase (NTE) is an integral membrane protein found predominantly in neurones. Covalent modification of NTE's active site serine, by certain organophosphates (OPs), results in a neurodegenerative syndrome. NTE's physiological substrate is unknown and its catalytic activity does not seem to be vital in the adult animal.;The enzyme domain of human NTE (residues 727-1216), called NEST, was expressed in E. coli and reacted with a carboxyl ester substrate and OP inhibitors the same as native NTE. During purification catalytic activity was lost, but was restored by reconstitution into the liposomes. Site-directed mutagenesis revealed that S966, D960 and D1086 were critical for catalysis. Mutation of two histidines, H860 and H885, also resulted in a loss of activity. Reacting NEST with [3H]diisopropylfluorophosphate, confirmed S966 as the active site serine and showed that an isopropyl group is transferred to an aspartate residue.;Standard hydropathy analysis predicts no membrane-spanning helices in NEST; however, biochemical evidence indicated that NEST is an integral membrane protein. TMpred analysis, on the other hand, predicts three transmembrane helices (TM2-4), with S966 at the centre of TM4. For S966 to be located within the membrane, TM4 would need to line the lumen of an aqueous pore to allow access of water for catalysis. Patch clamp studies on NEST-containing giant liposomes indicated that NEST forms a pore in vitro, while other experiments demonstrated that NEST monomers are catalytically active: this raises the possibility that NEST forms a -barrel structure in the membrane.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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