O‐Glycosylation and stability

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Abstract
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 1H 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.