O-GLYCOSYLATION AND STABILITY - UNFOLDING OF GLUCOAMYLASE INDUCED BY HEAT AND GUANIDINE-HYDROCHLORIDE

We have examined the stabilities of the catalytic and binding domains of glucoamylase 1 from Aspergillus niger and how these stabilities are affected by the O-glycosylated linker glycopeptide which separates the domains. On heating, the catalytic domain unfolds irreversibly, whereas the binding domain unfolds reversibly as shown by differential scanning calorimetry and by H-1 NMR. The stability of three functional peptides, derived from glucoamylase 1, containing the binding domain alone and with 10 or 38 residues of the linker glycopeptide [Williamson, G., Belshaw, N.J. and Williamson, M. (1992) Biochem. J. 282, 423- 428] was examined. Refolding in each case was reversible after thermal or chemical denaturation. Beta-Cyclodextrin stabilised the binding domain by the same amount when it was part of glucoamylase 1 or an isolated domain. The thermal stability of the catalytic domain was not affected by the binding domain; however, the catalytic domain increased the melting temperature of the binding domain. Furthermore, the linker glycopeptide stabilised the binding domain against reversible thermal and chemical denaturation by about 10 kJ/mol, but only a portion of the O-glycosylated residues were required for stabilisation. On a simple molecular mass basis, the linker glycopeptide does not contribute as much as expected to the denaturational enthalpy of glucoamylase 1 and, in addition, shows only a small conformational change on chemical or thermal denaturation; this supports an extended structure for the linker. The results demonstrate that the unfolding pathway of glucoamylase 1 depends on the concentration of beta-cyclodextrin and that the presence of the catalytic domain and/or the linker glycopeptide stabilises the binding domain.