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Secundo F., Russo C., Giordano A., Carrea G., Rossi M., Raia C.A. (2005)
Temperature-induced conformational change at the catalytic site of Sulfolobus solfataricus alcohol dehydrogenase highlighted by Asn249Tyr substitution. A hydrogen/deuterium exchange, kinetic, and fluorescence quenching study
in Biochemistry (Easton)
"^^rdf:HTML ; pubblicazioni:autori "Secundo F., Russo C., Giordano A., Carrea G., Rossi M., Raia C.A."^^xsd:string ; pubblicazioni:paginaInizio "11040"^^xsd:string ; pubblicazioni:paginaFine "11048"^^xsd:string ; pubblicazioni:numeroVolume "44"^^xsd:string . @prefix ns12: . prodotto:ID169118 pubblicazioni:rivista ns12:ID392889 ; skos:note "ISI Web of Science (WOS)"^^xsd:string ; pubblicazioni:titolo "Temperature-induced conformational change at the catalytic site of Sulfolobus solfataricus alcohol dehydrogenase highlighted by Asn249Tyr substitution. A hydrogen/deuterium exchange, kinetic, and fluorescence quenching study"^^xsd:string ; prodottidellaricerca:abstract "A combination of hydrogen/deuterium exchange, fluorescence quenching, and kinetic studies was used to acquire experimental evidence for the crystallographically hypothesized increase in local flexibility which occurs in thermophilic NAD(+)-dependent Sulfolobus solfataricus alcohol dehydrogenase (SsADH) upon substitution Asn249Tyr. The substitution, located at the adenine-binding site, proved to decrease the affinity for both coenzyme and substrate, rendering the mutant enzyme 6-fold more active when compared to the wild-type enzyme [Esposito et al. (2003) FEBS Lett. 539, 14-18]. The amide H/D exchange data show that the wild-type and mutant enzymes have similar global flexibility at 22 and 60 degrees C. However, the temperature dependence of the Stern-Volmer constant determined by acrylamide quenching shows that the increase in temperature affects the local flexibility differently, since the Ksv increment is significantly higher for the wild-type than for the mutant enzyme over the range 18-45 degrees C. Interestingly, the corresponding van't Hoff plot (log Ksv vs 1/T) proves nonlinear for the apo and holo wild-type and apo mutant enzymes, with a break at approximate to 45 degrees C in all three cases due to a conformational change affecting the tryptophan microenvironment experienced by the quencher molecules. The Arrhenius and van't Hoff plots derived from the k(cat) and K-M thermodependence measured with cyclohexanol and NAD+ at different temperatures display an abrupt change of slope at 45-50 degrees C. This proves more pronounced in the case of the mutant enzyme compared to the wild-type enzyme due to a conformational change in the structure rather than to an overlapping of two or more rate-limiting reaction steps with different temperature dependencies of their rate constants. Three-dimensional analysis indicates that the observed conformational change induced by temperature is associated with the flexible loops directly involved in the substrate and coenzyme binding." ; prodottidellaricerca:prodottoDi modulo:ID2129 , modulo:ID2531 , istituto:CDS007 , istituto:CDS021 ; pubblicazioni:autoreCNR unitaDiPersonaleInterno:MATRICOLA18403 , unitaDiPersonaleInterno:MATRICOLA19348 , unitaDiPersonaleInterno:MATRICOLA3831 , unitaDiPersonaleEsterno:ID6206 . ns12:ID392889 pubblicazioni:rivistaDi prodotto:ID169118 .