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Effect of a Thr81→ Ala Mutation at the Subunit Effect of a T81A mutation at the subunit interface on catalytic properties of alkaline phosphatase from Escherichia coli (CROSBI ID 117914)

Prilog u časopisu | izvorni znanstveni rad | međunarodna recenzija

Orhanović, Stjepan ; Bučević-Popović, Viljemka ; Pavela-Vrančič, Maja ; Vujaklija, Dušica ; Gamulin, Vera Effect of a Thr81→ Ala Mutation at the Subunit Effect of a T81A mutation at the subunit interface on catalytic properties of alkaline phosphatase from Escherichia coli // International journal of biological macromolecules, 40 (2006), 1; 54-58. doi: 10.1016/j.ijbiomac.2006.06.008

Podaci o odgovornosti

Orhanović, Stjepan ; Bučević-Popović, Viljemka ; Pavela-Vrančič, Maja ; Vujaklija, Dušica ; Gamulin, Vera

engleski

Effect of a Thr81→ Ala Mutation at the Subunit Effect of a T81A mutation at the subunit interface on catalytic properties of alkaline phosphatase from Escherichia coli

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 conformationally and kinetically non- equivalent subunits. The proposed model, explaining the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions [Orhanović S., Pavela-Vrančič M. (2003) Eur. J. Biochem. 270, 4356-4364]. The significance of interactions at the subunit interface and the involvement of the β -pleated sheet stretching from underneath the active site to the subunit surface, in the catalytic mechanism, has been probed by site directed mutagenesis. The mutant APase, carrying alanine in place of Thr81, was analyzed in comparison to the wild type protein. The T81A mutant proved to be more sensitive to thermal denaturation, and undergoes irreversible dissociation to monomers upon depletion of metal ions by EDTA. The affinity of T81A for the substrate and Pi was slightly reduced in 2A2M1P buffer at pH 10.5. In 1 M Tris/HCl, pH 8, the turnover number kcat of the T81A mutant was reduced compared to the wild type enzyme, indicating that the mutation, affecting interactions between β -pleated sheets at the subunit interface, influences the rate determining step at pH 8. The T81→ A mutation, introduced at the subunit interface, affected both structural stability of the protein and its kinetic properties, emphasizing the importance of subunit interactions in the catalytic process.

alkaline phosphatase ; kinetic properties ; conformational changes ; subunit interactions ; enzyme asymmetry

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Podaci o izdanju

40 (1)

2006.

54-58

objavljeno

0141-8130

1879-0003

10.1016/j.ijbiomac.2006.06.008

Povezanost rada

Biologija, Kemija

Poveznice
Indeksiranost