Williamson G, Belshaw N J, Noel T R, Ring S G, Williamson M P
AFRC Institute of Food Research, Norwich Laboratory, Norwich Research Park, England.
Eur J Biochem. 1992 Jul 15;207(2):661-70. doi: 10.1111/j.1432-1033.1992.tb17093.x.
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.
我们研究了黑曲霉葡糖淀粉酶1催化结构域和结合结构域的稳定性，以及连接这两个结构域的O-糖基化连接糖肽对这些稳定性的影响。通过差示扫描量热法和1H NMR表明，加热时催化结构域不可逆地展开，而结合结构域可逆地展开。我们研究了源自葡糖淀粉酶1的三种功能肽的稳定性，这些肽分别单独包含结合结构域以及带有连接糖肽的10个或38个残基[威廉姆森，G.，贝尔肖，N.J.和威廉姆森，M.（1992年）《生物化学杂志》282卷，423 - 428页]。在每种情况下，热变性或化学变性后复性都是可逆的。当β-环糊精是葡糖淀粉酶1的一部分或分离的结构域时，它对结合结构域的稳定作用相同。催化结构域的热稳定性不受结合结构域的影响；然而，催化结构域提高了结合结构域的解链温度。此外，连接糖肽使结合结构域对可逆热变性和化学变性的稳定性提高了约10 kJ/mol，但稳定作用仅需要一部分O-糖基化残基。基于简单的分子量，连接糖肽对葡糖淀粉酶1变性焓的贡献不如预期的大，并且在化学或热变性时仅显示出小的构象变化；这支持连接糖肽具有伸展结构。结果表明，葡糖淀粉酶1的展开途径取决于β-环糊精的浓度，并且催化结构域和/或连接糖肽的存在使结合结构域稳定。
