Thermodynamics of reversible and irreversible unfolding and domain interactions of glucoamylase from Aspergillus niger studied by differential scanning and isothermal titration calorimetry

The stability of three forms of glucoamylase from Aspergillus niger has bee n investigated by differential scanning and isothermal titration calorimetr y: Glucoamylase 1 (GA1), which consists of a catalytic domain and a starch- binding domain (SBD) connected by a heavily O-glycosylated linker region; g lucoamylase 2 (GA2), which lacks SBD; and a proteolytically cleaved glucoam ylase (GACD), which contains the catalytic domain and part of the linker re gion. The structures of the catalytic domain with part of the linker region and of SBD are known from crystallography and NMR, respectively, but the p recise spatial arrangement of the two domains in GA1 is unknown. To investi gate the stability of the three glucoamylase forms, we unfolded the enzymes thermally by differential scanning calorimetry (DSC). Aggregation occurs u pon heating GA1 and GA2 at pH values between 2.5 and 5.0, whereas no aggreg ation is observed at higher pH (5.5-7.5). At all pH values, the catalytic d omain 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 th e catalytic domain of all glucoamylase forms seems to follow an irreversibl e one-step mechanism with no observable reversible intermediates on the exp erimental 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 pe aks of the DSC profile to the domains at pH 7.5 is achieved by using two di fferent ligands: Acarbose, a very strong inhibitor that binds exclusively t o the catalytic domain, and P-cyclodextrin, a small starch analogue of whic h 2 molecules bind solely to the two binding sites present in SBD. Differen ces are seen in the unfolding processes of GA1 and GA2 since the former unf olds with one peak at all pH values, while the calorimetric trace of the la tter can be resolved into more peaks depending on pH and the chemical compo sition 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 b efore 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 t he conclusion that the structure of the GA2 molecule has a larger cooperati ve unfolding unit and is less stable than the structures of GA1 and GACD an d that the C-terminal part of the linker region has a destabilizing effect on the catalytic domain.