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Pregled bibliografske jedinice broj: 218986

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


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 (međunarodna recenzija, članak, znanstveni)


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Naslov
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

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

Izvornik
International Journal of Biological Macromolecules (0141-8130) 40 (2006), 1; 54-58

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

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

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 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.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija



POVEZANOST RADA


Projekt / tema
0177150
0098072

Ustanove
Institut "Ruđer Bošković", Zagreb

Citiraj ovu publikaciju

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 (međunarodna recenzija, članak, znanstveni)
Orhanović, S., Bučević-Popović, V., Pavela-Vrančič, M., Vujaklija, D. & Gamulin, V. (2006) 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 (1), 54-58 doi:10.1016/j.ijbiomac.2006.06.008.
@article{article, year = {2006}, pages = {54-58}, DOI = {10.1016/j.ijbiomac.2006.06.008}, keywords = {alkaline phosphatase, kinetic properties, conformational changes, subunit interactions, enzyme asymmetry}, journal = {International Journal of Biological Macromolecules}, doi = {10.1016/j.ijbiomac.2006.06.008}, volume = {40}, number = {1}, issn = {0141-8130}, title = {Effect of a Thr81 and \#8594; Ala Mutation at the Subunit Effect of a T81A mutation at the subunit interface on catalytic properties of alkaline phosphatase from Escherichia coli}, keyword = {alkaline phosphatase, kinetic properties, conformational changes, subunit interactions, enzyme asymmetry} }

Časopis indeksira:


  • Current Contents Connect (CCC)
  • Web of Science Core Collection (WoSCC)
    • Science Citation Index Expanded (SCI-EXP)
    • SCI-EXP, SSCI i/ili A&HCI
  • Scopus
  • MEDLINE


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