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Titolo:
Probing the acyl binding site of acetylcholinesterase by protein engineering
Autore:
Pleiss, J; Mionetto, N; Schmid, RD;
Indirizzi:
Univ Stuttgart, Inst Tech Biochem, D-70569 Stuttgart, Germany Univ Stuttgart Stuttgart Germany D-70569 hem, D-70569 Stuttgart, Germany
Titolo Testata:
JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
fascicolo: 3, volume: 6, anno: 1999,
pagine: 287 - 296
SICI:
1381-1177(19990311)6:3<287:PTABSO>2.0.ZU;2-M
Fonte:
ISI
Lingua:
ENG
Soggetto:
SUBSTRATE-SPECIFICITY; ANIONIC SITE; DATA-BANK; CHOLINESTERASES; BUTYRYLCHOLINESTERASE; INHIBITION; COMPLEX; LIPASE; MUTAGENESIS; EXPRESSION;
Keywords:
protein engineering; mutation; acetylcholinesterase; inhibitors;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Physical, Chemical & Earth Sciences
Citazioni:
37
Recensione:
Indirizzi per estratti:
Indirizzo: Schmid, RD Univnytuttgart, Inst Tech Biochem, Allmandring 31, D-70569 Stuttgart, Germa Univ Stuttgart Allmandring 31 Stuttgart Germany D-70569 Germa
Citazione:
J. Pleiss et al., "Probing the acyl binding site of acetylcholinesterase by protein engineering", J MOL CAT B, 6(3), 1999, pp. 287-296

Abstract

Recombinant acetylcholinesterase from rat brain and two mutants were studied for their hydrolytic activity toward acetyland butyrylthiocholine substrates and for their sensitivity toward organophosphate and carbamate inhibitors. Both mutants, a point mutant where F295 was replaced by leucine, and asecond mutant where loop PQES was replaced by SG, were designed for increased size of the acyl binding pocket. Wild type and mutant enzymes were expressed in baculovirus-infected insect cells and biochemically characterized. As expected, wild type rat brain acetylcholinesterase hydrolyzed acetylthiocholine, but not butyrylthiocholine. Sensitivity toward small- and medium-sized organophosphate inhibitors like paraoxon-methyl and paraoxon-ethyl was comparable, but bulky organophosphates like ethoprophos were less efficient inhibitors. This tendency applied to carbamates as well, since small carbamoyl moieties like carbofuran and aldicarb were stronger inhibitors than furathiocarb which features a bulky carbamoyl moiety. In contrast to wild type enzyme, both mutants were capable of hydrolyzing butyrylthiocholine. However, k(cat)/K-m toward acetylthiocholine of the F295L mutant was reduced if compared to the wild type enzyme. All five organophosphate and three carbamate inhibitors inhibited mutant F295L more efficiently than the wild type enzyme. (C) 1999 Elsevier Science B.V. All rights reserved.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/12/20 alle ore 00:41:46