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.
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.
Secundo
Francesco
F
Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Via M. Bianco 9, I-20131 Milano, Italy.
Russo
Consiglia
C
Giordano
Antonietta
A
Carrea
Giacomo
G
Rossi
Mosè
M
Raia
Carlo A
CA
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D003903:Deuterium / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence / Q000379:methods; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics
D009243:NAD; D003903:Deuterium; D000426:Alcohol Dehydrogenase