Kinetic and structural properties of a mutant form of alkaline phosphatase from E. coli. (CROSBI ID 512383)
Prilog sa skupa u zborniku | sažetak izlaganja sa skupa
Podaci o odgovornosti
Stjepan Orhanović, Viljemka Bučević- Popović, Maja Pavela-Vrančič
engleski
Kinetic and structural properties of a mutant form of alkaline phosphatase from E. coli.
Alkaline phosphatase (APase, E.C. 3.1.3.1) is a non-specific phosphomonoesterase catalysing the hydrolysis of a large number of phosphomonoesters. Most unresolved questions, relating to the catalytic mechanism of alkaline phosphatase concern the influence of conformational changes and allosteric interactions on the catalytic efficiency. Rigid segments in the polypeptide structure supposedly accomplish transfer of conformational information, between the active sites, across the subunit interface [1]. 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[2]. 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 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. Catalytic constant was reduced twofold in 1 M Tris/HCl, pH 8. 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.
Alkaline phosphatase: Kinetic properties; Conformational changes; Subunit interactions; Enzyme asymmetry.
nije evidentirano
nije evidentirano
nije evidentirano
nije evidentirano
nije evidentirano
nije evidentirano
Podaci o prilogu
83-83.
2005.
nije evidentirano
objavljeno
9536894238
Podaci o matičnoj publikaciji
XIX. Hrvatski skup kemičara i kemijskih inženjera - knjiga sažetaka
Rapić, Vladimir ; Rogošić, Marko
Zagreb: Hrvatsko društvo Kemija u industriji
Podaci o skupu
XIX Hrvatski skup kemičara i kemijskih inženjera
poster
24.04.2005-27.04.2005
Opatija, Hrvatska