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
| Autor: | Francesco Secundo, Mosè Rossi, Giacomo Carrea, Consiglia Russo, Antonietta Giordano, Carlo A. Raia |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Conformational change
Protein Folding Hot Temperature Stereochemistry ved/biology.organism_classification_rank.species Biochemistry symbols.namesake Coenzyme binding Point Mutation Arrhenius equation Quenching (fluorescence) Binding Sites ved/biology Chemistry Sulfolobus solfataricus Alcohol Dehydrogenase Substrate (chemistry) Deuterium NAD Protein Structure Tertiary Crystallography Kinetics Spectrometry Fluorescence Amino Acid Substitution symbols Hydrogen–deuterium exchange NAD+ kinase Protein Binding |
| Zdroj: | Biochemistry. 44(33) |
| ISSN: | 0006-2960 |
| Popis: | 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 K(SV) 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 K(SV) vs 1/T) proves nonlinear for the apo and holo wild-type and apo mutant enzymes, with a break at approximately 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|