Chen L, Coutinho P M, Nikolov Z, Ford C
Department of Zoology, Iowa State University, Ames 50011, USA.
Protein Eng. 1995 Oct;8(10):1049-55. doi: 10.1093/protein/8.10.1049.
The large form of glucoamylase (GAI) from Aspergillus awamori (EC 3.2.1.3) binds strongly to native granular starch, whereas a truncated form (GAII) which lacks 103 C-terminal residues, does not. This C-terminal region, conserved among fungal glucoamylases and other starch-degrading enzymes, is part of an independent starch-binding domain (SBD). To investigate the SBD boundaries and the function of conserved residues in two putative substrate-binding sites, five gluco-amylase mutants were constructed with extensive deletions in this region for expression in Saccharomyces cerevisiae. Progressive loss of both starch-binding and starch-hydrolytic activity occurred upon removal of eight and 25 C-terminal amino acid residues, or 21 and 52 residues close to the N-terminus, confirming the requirement for the entire region in formation of a functional SBD. C-terminal deletions strongly impaired SBD function, suggesting a more important role for one of the putative binding sites. A GAII phenocopy showed a nearly complete loss of starch-binding and starch-hydrolytic activity. The deletions did not affect enzyme activity on soluble starch or thermo-stability of the enzyme, confirming the independence of the catalytic domain from the SBD.
泡盛曲霉（EC 3.2.1.3）的大型糖化酶（GAI）与天然颗粒淀粉紧密结合，而缺少103个C末端残基的截短形式（GAII）则不然。这个在真菌糖化酶和其他淀粉降解酶中保守的C末端区域，是一个独立淀粉结合结构域（SBD）的一部分。为了研究SBD的边界以及两个假定底物结合位点中保守残基的功能，构建了五个在该区域有大量缺失的糖化酶突变体，用于在酿酒酵母中表达。去除8个和25个C末端氨基酸残基，或靠近N末端的21个和52个残基后，淀粉结合活性和淀粉水解活性逐渐丧失，这证实了形成功能性SBD需要整个区域。C末端缺失严重损害了SBD功能，表明其中一个假定结合位点的作用更为重要。一个GAII表型显示淀粉结合活性和淀粉水解活性几乎完全丧失。这些缺失不影响酶对可溶性淀粉的活性或酶的热稳定性，证实了催化结构域与SBD的独立性。
