Williamson G, Belshaw N J, Williamson M P
AFRC Institute of Food Research, Norwich Laboratory, U.K.
Biochem J. 1992 Mar 1;282 ( Pt 2)(Pt 2):423-8. doi: 10.1042/bj2820423.
Functional peptides have been produced by proteolysis of glucoamylase (glucan 1,4-alpha-glucosidase; EC 3.2.1.3) from Aspergillus niger and purified by affinity chromatography, gel filtration and two ion-exchange-chromatography steps. The peptides correspond to residues 499-616 and 509-616 of the original glucoamylase molecule. Together with G1C (residues 471-616 from glucoamylase 1) [Belshaw & Williamson (1990) FEBS Lett. 269, 350-353], the three peptides all contain the C-terminal domain (residues 509-616) but, in addition, contain different proportions of the O-glycosylated region. The properties of these peptides have been compared to define the function of the O-linked oligosaccharides in this protein. The O-glycosylated region plays only a minor role in binding to hydrogen-bond ordered starch. The difference between the apparent free energy (delta G) for binding between the non-glycosylated C-terminal domain (-26.0 kJ/mol) and the C-terminal domain containing the fully O-glycosylated region (-25.0 kJ/mol) is only 1.0 kJ/mol. Binding to beta-cyclodextrin suggests that even this difference may reflect a small conformational change in the C-terminal domain rather than a direct effect of the O-linked sugars. The c.d. spectrum of the O-glycosylated region is deduced by comparison of the three peptides and is predominantly that of a random-coil structure. Two-dimensional n.m.r. spectra of glucoamylase and of the glycosylated peptide 499-616 show that the binding domain is more mobile than the catalytic domain and that its mobility is further increased on removal of the catalytic domain. The O-glycosylated region is more mobile still, and there is a marked increase in its mobility on removal of the catalytic domain. The O-glycosylated region in the intact protein can therefore be envisaged as a semi-rigid rod. The results show that a major function of O-glycosylation in glucoamylase 1 is to provide an extended peptide backbone and hence a fixed distance in linking the catalytic and binding domains. It does not in itself significantly increase the binding affinity for starch.
通过黑曲霉葡糖淀粉酶（葡聚糖1,4-α-葡糖苷酶；EC 3.2.1.3）的蛋白水解作用产生了功能肽，并通过亲和色谱、凝胶过滤和两步离子交换色谱步骤进行了纯化。这些肽对应于原始葡糖淀粉酶分子的499 - 616位残基和509 - 616位残基。与G1C（葡糖淀粉酶1的471 - 616位残基）[Belshaw & Williamson (1990) FEBS Lett. 269, 350 - 353]一起，这三种肽都包含C末端结构域（509 - 616位残基），但此外，还包含不同比例的O-糖基化区域。已对这些肽的特性进行了比较，以确定该蛋白中O-连接寡糖的功能。O-糖基化区域在与氢键有序淀粉的结合中仅起次要作用。非糖基化C末端结构域（-26.0 kJ/mol）与包含完全O-糖基化区域的C末端结构域（-25.0 kJ/mol）之间结合的表观自由能（ΔG）差异仅为1.0 kJ/mol。与β-环糊精的结合表明，即使这种差异也可能反映了C末端结构域的微小构象变化，而不是O-连接糖的直接作用。通过对这三种肽的比较推导了O-糖基化区域 的圆二色光谱，其主要为无规卷曲结构的光谱。葡糖淀粉酶和糖基化肽499 - 616的二维核磁共振光谱表明，结合结构域比催化结构域更具流动性，并且在去除催化结构域后其流动性进一步增加。O-糖基化区域的流动性更大，并且在去除催化结构域后其流动性显著增加。因此，完整蛋白中的O-糖基化区域可被设想为一个半刚性杆。结果表明，葡糖淀粉酶1中O-糖基化的主要功能是提供一个延伸的肽主链，从而在连接催化结构域和结合结构域时提供一个固定的距离。它本身并不会显著增加对淀粉的结合亲和力。
