Naslov
Site-directed mutagenesis of acetylcholinesterase: a tool for studying structure/function relationship

Autori
Kovarik, Zrinka ; Radić, Zoran ; Taylor, Palmer

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Knjiga sažetaka / 2. znanstveni simpozij s međunarodnim sudjelovanjem: 45 godina molekularne biologije u Hrvatskoj i 50 godina dvostruke uzvojnice / Ambriović Ristov, Andreja ; Brozović, Anamaria - Zagreb : Farmaceutsko-biokemijski fakultet Sveučilišta u Zagrebu, 2003

Skup
2. znanstveni simpozij s međunarodnim sudjelovanjem: 45 godina molekularne biologije u Hrvatskoj i 50 godina dvostruke uzvojnice

Mjesto i datum
Zagreb, Hrvatska, 20.11.2003. - 21.11.2003

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
site-directed mutagenesis; acetylcholinesterase

Sažetak
Seven site-directed mutants of mouse acetylcholinesterase (AChE ; EC 3.1.1.8) were constructed in order to examine the structural bases of mechanism of AChE interaction with inhibitors. Mutants were selected based on location in the AChE active site and on possible role in AChE interactions. The mutations combined substitutions in the choline binding site (Y337A, F338A) with the acyl pocket (F295L, F295A, F297I, F297A) and with the N-terminal residue to the catalytic serine (E202Q). A cDNA encoding mouse AChE truncated at position 548, which yields a monomeric form of the enzyme, was used in mutagenesis as the wild type cDNA. Mutant DNA was generated by PCR mediated mutagenesis while multiple mutants were generated by subcloning of DNA fragments containing single mutations into the mammalian expression vector, pCDNA3 or pRCCMV. Human embryonic kidney cells were transfected, and stable transfectants were generated by selecting transfected HEK cells with G418. Harvests of the media containing the mutant AChE were subjected to purification by affinity chromatography using procainamide affinity resin and decamethonium as elutant. After dialysis of the purified mutant, levels of purity were determined by SDS-PAGE using the endoglycosidase, PGNase F. The mutations affected the catalysis by increasing Km and decreasing kcat, by an order of magnitude or less, which is a relatively small change compared to the catalytic potential of AChE. Consequently, generated mutants are catalytically efficient enzymes, and can be used as a tool for studing AChE structure/function relationships in the interaction with inhibitors.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

POVEZANOST RADA