Thermodynamics of Reversible and Irreversible Unfolding and Domain Interactions of Glucoamylase from Aspergillus niger Studied by Differential Scanning and Isothermal Titration Calorimetry
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- 作者：Trine Christensen ; Birte Svensson ; and Bent W. Sigurskjold
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：May 11, 1999
- 年：1999
- 卷：38
- 期：19
- 页码：6300 - 6310
- 全文大小：108K
- 年卷期：v.38,no.19(May 11, 1999)
- ISSN：1520-4995
文摘
The stability of three forms of glucoamylase from Aspergillus niger has been investigated bydifferential scanning and isothermal titration calorimetry: Glucoamylase 1 (GA1), which consists of acatalytic domain and a starch-binding domain (SBD) connected by a heavily O-glycosylated linker region;glucoamylase 2 (GA2), which lacks SBD; and a proteolytically cleaved glucoamylase (GACD), whichcontains the catalytic domain and part of the linker region. The structures of the catalytic domain withpart of the linker region and of SBD are known from crystallography and NMR, respectively, but theprecise spatial arrangement of the two domains in GA1 is unknown. To investigate the stability of thethree glucoamylase forms, we unfolded the enzymes thermally by differential scanning calorimetry (DSC).Aggregation occurs upon heating GA1 and GA2 at pH values between 2.5 and 5.0, whereas no aggregationis observed at higher pH (5.5-7.5). At all pH values, the catalytic domain of GA1 and GA2 unfoldsirreversibly, while SBD unfolds reversibly in the pH range 5.5-7.5 where aggregation does not occur.The unfolding of the catalytic domain of all glucoamylase forms seems to follow an irreversible one-stepmechanism with no observable reversible intermediates on the experimental time scale. SBD of GA1unfolds reversibly, and the ratio between the van't Hoff and calorimetric enthalpies is 1.4 ± 0.1. Assignmentof peaks of the DSC profile to the domains at pH 7.5 is achieved by using two different ligands: Acarbose,a very strong inhibitor that binds exclusively to the catalytic domain, and 
-cyclodextrin, a small starchanalogue of which 2 molecules bind solely to the two binding sites present in SBD. Differences are seenin the unfolding processes of GA1 and GA2 since the former unfolds with one peak at all pH values,while the calorimetric trace of the latter can be resolved into more peaks depending on pH and the chemicalcomposition of the buffers. In general, peaks corresponding to unfolding of GA2 are more complex thanthe peaks of GA1 and GACD. Some part of GA2 unfolds before the rest of the molecule which maycorrespond to the linker region or a particular early unfolding part of the catalytic domain. This leads tothe conclusion that the structure of the GA2 molecule has a larger cooperative unfolding unit and is lessstable than the structures of GA1 and GACD and that the C-terminal part of the linker region has adestabilizing effect on the catalytic domain.
-cyclodextrin, a small starchanalogue of which 2 molecules bind solely to the two binding sites present in SBD. Differences are seenin the unfolding processes of GA1 and GA2 since the former unfolds with one peak at all pH values,while the calorimetric trace of the latter can be resolved into more peaks depending on pH and the chemicalcomposition of the buffers. In general, peaks corresponding to unfolding of GA2 are more complex thanthe peaks of GA1 and GACD. Some part of GA2 unfolds before the rest of the molecule which maycorrespond to the linker region or a particular early unfolding part of the catalytic domain. This leads tothe conclusion that the structure of the GA2 molecule has a larger cooperative unfolding unit and is lessstable than the structures of GA1 and GACD and that the C-terminal part of the linker region has adestabilizing effect on the catalytic domain.