Temperature-Induced Denaturation and Renaturation of Triosephosphate Isomerase from Saccharomyces cerevisiae: Evidence of Dimerization Coupled to Refolding of the Thermally Unfolded Protein
详细信息 查看全文
- 作者：Claudia G. Bení ; tez-Cardoza ; Arturo Rojo-Domí ; nguez ; and André ; s Herná ; ndez-Arana
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：July 31, 2001
- 年：2001
- 卷：40
- 期：30
- 页码：9049 - 9058
- 全文大小：114K
- 年卷期：v.40,no.30(July 31, 2001)
- ISSN：1520-4995
文摘
The thermal denaturation of the dimeric enzyme triosephosphate isomerase (TIM) fromSaccharomyces cerevisiae was studied by spectroscopic and calorimetric methods. At low proteinconcentration the structural transition proved to be reversible in thermal scannings conducted at a rategreater than 1.0 
C min-1. Under these conditions, however, the denaturation-renaturation cycle exhibitedmarked hysteresis. The use of lower scanning rates lead to pronounced irreversibility. Kinetic studiesindicated that denaturation of the enzyme likely consists of an initial first-order reaction that forms thermallyunfolded (U) TIM, followed by irreversibility-inducing reactions which are probably linked to aggregationof the unfolded protein. As judged from CD measurements, U possesses residual secondary structure butlacks most of the tertiary interactions present in native TIM. Furthermore, the large increment in heatcapacity upon denaturation suggests that extensive exposure of surface area occurs when U is formed.Above 63 C, reactions leading to irreversibility were much slower than the unfolding process; as a result,U was sufficiently long-lived as to allow an investigation of its refolding kinetics. We found that Utransforms into nativelike TIM through a second-order reaction in which association is coupled to theregain of secondary structure. The rate constants for unfolding and refolding of TIM displayed temperaturedependences resembling those reported for monomeric proteins but with considerably larger activationenthalpies. Such large temperature dependences seem to be determinant for the occurrence of kineticallycontrolled transitions and thus constitute a simple explanation for the hysteresis observed in thermalscannings.
C min-1. Under these conditions, however, the denaturation-renaturation cycle exhibitedmarked hysteresis. The use of lower scanning rates lead to pronounced irreversibility. Kinetic studiesindicated that denaturation of the enzyme likely consists of an initial first-order reaction that forms thermallyunfolded (U) TIM, followed by irreversibility-inducing reactions which are probably linked to aggregationof the unfolded protein. As judged from CD measurements, U possesses residual secondary structure butlacks most of the tertiary interactions present in native TIM. Furthermore, the large increment in heatcapacity upon denaturation suggests that extensive exposure of surface area occurs when U is formed.Above 63 C, reactions leading to irreversibility were much slower than the unfolding process; as a result,U was sufficiently long-lived as to allow an investigation of its refolding kinetics. We found that Utransforms into nativelike TIM through a second-order reaction in which association is coupled to theregain of secondary structure. The rate constants for unfolding and refolding of TIM displayed temperaturedependences resembling those reported for monomeric proteins but with considerably larger activationenthalpies. Such large temperature dependences seem to be determinant for the occurrence of kineticallycontrolled transitions and thus constitute a simple explanation for the hysteresis observed in thermalscannings.