Naslov
Purification and characterisation of a mutant form of alkaline phosphatase from E. coli

Autori
Orhanović, Stjepan ; Bučević-Popović, Viljemka ; Pavela-Vrančič, Maja

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

Izvornik
Congress of the Croatian society of biochemistry and molecular biology with international participation - Book of Abstracts / Dumić, Jerka - Zagreb : Farmaceutsko-biokemijski fakultet Sveučilišta u Zagrebu, 2004

Skup
Congress of the Croatian society of biochemistry and molecular biology with international participation

Mjesto i datum
HOC Bjelolasica, Hrvatska, 30.09.2004. - 02.10.2004

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
alkaline phoshatase; site-directed mutagenesis; conformational changes; allosteric interactions

Sažetak
Although alkaline phosphatase (APase) from E. coli crystallizes as a symmetric dimer, it displays deviations from Michaelis-Menten kinetics, supported by a model describing a dimeric enzyme with unequal subunits. The proposed model, describing the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions. Rigid parts in the polypeptide structure supposedly accomplish transfer of conformational information between the active sites across the subunit interface. The importance of the subunit interface and the β -pleated sheet, stretching from underneath an active site to the subunit surface, in the catalytic mechanism has been probed by site-directed mutagenesis. The mutation replacing Thr-81 with Ala was introduced into the APase gene. Thr-81 is located within the β -pleated sheet at the contact surface between the subunits, and forms hydrogen bonds with the analogous Thr-81 from the adjacent subunit. The mutant protein was purified and compared with the wild type APase. Stability of the protein was assessed by thermal and urea denaturation. The affinity for metal ligands and their influence on the quaternary structure was examined by monitoring the inactivation of the enzyme in the presence of EDTA. Kinetic properties have been determined in 1 M Tris/HCl, pH 8 and in 0.35 M 2A2M1P, pH 10.5. The mutant enzyme proved to be more sensitive to inactivation with EDTA and to thermal denaturation. Following dialysis in the presence of EDTA the mutant enzyme dissociated into subunits contrary to the wild-type protein that maintains its dimeric structure. The activity of the mutant protein could not be reconstituted upon addition of metal ions. The affinity of the mutant enzyme for the substrate and for inorganic phosphate was reduced in 2A2M1P buffer at pH 10.5. The mutation introduced at the subunit interface affected both structural stability of the protein and its kinetic properties emphasising the importance of subunit interaction in the catalytic mechanism.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA