Temperature-Induced Conformational Change at the Catalytic Site of Sulfolobus solfataricus Alcohol Dehydrogenase Highlighted by Asn249Tyr Substitution. A Hydrogen/Deuterium Exchange, Kinetic, a
详细信息 查看全文
- 作者：Francesco Secundo ; Consiglia Russo ; Antonietta Giordano ; Giacomo Carrea ; Mosè ; Rossi ; and Carlo A. Raia
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：August 23, 2005
- 年：2005
- 卷：44
- 期：33
- 页码：11040 - 11048
- 全文大小：298K
- 年卷期：v.44,no.33(August 23, 2005)
- ISSN：1520-4995
文摘
A combination of hydrogen/deuterium exchange, fluorescence quenching, and kinetic studieswas used to acquire experimental evidence for the crystallographically hypothesized increase in localflexibility which occurs in thermophilic NAD+-dependent Sulfolobus solfataricus alcohol dehydrogenase(SsADH) upon substitution Asn249Tyr. The substitution, located at the adenine-binding site, proved todecrease the affinity for both coenzyme and substrate, rendering the mutant enzyme 6-fold more activewhen compared to the wild-type enzyme [Esposito et al. (2003) FEBS Lett. 539, 14-18]. The amide H/Dexchange data show that the wild-type and mutant enzymes have similar global flexibility at 22 and 60
C. However, the temperature dependence of the Stern-Volmer constant determined by acrylamidequenching shows that the increase in temperature affects the local flexibility differently, since the KSVincrement is significantly higher for the wild-type than for the mutant enzyme over the range 18-45 C.Interestingly, the corresponding van't Hoff plot (log KSV vs 1/T) proves nonlinear for the apo and holowild-type and apo mutant enzymes, with a break at 
45 C in all three cases due to a conformationalchange affecting the tryptophan microenvironment experienced by the quencher molecules. The Arrheniusand van't Hoff plots derived from the kcat and KM thermodependence measured with cyclohexanol andNAD+ at different temperatures display an abrupt change of slope at 45-50 C. This proves morepronounced in the case of the mutant enzyme compared to the wild-type enzyme due to a conformationalchange in the structure rather than to an overlapping of two or more rate-limiting reaction steps withdifferent temperature dependencies of their rate constants. Three-dimensional analysis indicates that theobserved conformational change induced by temperature is associated with the flexible loops directlyinvolved in the substrate and coenzyme binding.
C. However, the temperature dependence of the Stern-Volmer constant determined by acrylamidequenching shows that the increase in temperature affects the local flexibility differently, since the KSVincrement is significantly higher for the wild-type than for the mutant enzyme over the range 18-45 C.Interestingly, the corresponding van't Hoff plot (log KSV vs 1/T) proves nonlinear for the apo and holowild-type and apo mutant enzymes, with a break at 45 C in all three cases due to a conformationalchange affecting the tryptophan microenvironment experienced by the quencher molecules. The Arrheniusand van't Hoff plots derived from the kcat and KM thermodependence measured with cyclohexanol andNAD+ at different temperatures display an abrupt change of slope at 45-50 C. This proves morepronounced in the case of the mutant enzyme compared to the wild-type enzyme due to a conformationalchange in the structure rather than to an overlapping of two or more rate-limiting reaction steps withdifferent temperature dependencies of their rate constants. Three-dimensional analysis indicates that theobserved conformational change induced by temperature is associated with the flexible loops directlyinvolved in the substrate and coenzyme binding.