Abstract:
:The stability of three forms of glucoamylase from Aspergillus niger has been investigated by differential scanning and isothermal titration calorimetry: Glucoamylase 1 (GA1), which consists of a catalytic 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), which contains the catalytic domain and part of the linker region. The structures of the catalytic domain with part of the linker region and of SBD are known from crystallography and NMR, respectively, but the precise spatial arrangement of the two domains in GA1 is unknown. To investigate the stability of the three 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 aggregation is observed at higher pH (5.5-7.5). At all pH values, the catalytic domain of GA1 and GA2 unfolds irreversibly, 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-step mechanism with no observable reversible intermediates on the experimental time scale. SBD of GA1 unfolds reversibly, and the ratio between the van't Hoff and calorimetric enthalpies is 1.4 +/- 0.1. Assignment of 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 beta-cyclodextrin, a small starch analogue of which 2 molecules bind solely to the two binding sites present in SBD. Differences are seen in 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 chemical composition of the buffers. In general, peaks corresponding to unfolding of GA2 are more complex than the peaks of GA1 and GACD. Some part of GA2 unfolds before the rest of the molecule which may correspond to the linker region or a particular early unfolding part of the catalytic domain. This leads to the conclusion that the structure of the GA2 molecule has a larger cooperative unfolding unit and is less stable than the structures of GA1 and GACD and that the C-terminal part of the linker region has a destabilizing effect on the catalytic domain.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Christensen T,Svensson B,Sigurskjold BWdoi
10.1021/bi990185qsubject
Has Abstractpub_date
1999-05-11 00:00:00pages
6300-10issue
19eissn
0006-2960issn
1520-4995pii
bi990185qjournal_volume
38pub_type
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