Oligosaccharide Binding to Barley α-Amylase 1*
Oligosaccharide Binding to Barley α-Amylase 1*
复制标题
DOI：
10.1074/jbc.m505515200
复制
发表时间：
2005-09
影响因子：
4.8
通讯作者：
X. Robert;R. Haser;H. Mori;B. Svensson;N. Aghajari
中科院分区：
文献类型：
--
作者：
X. Robert;R. Haser;H. Mori;B. Svensson;N. Aghajari
酶亚位点定位法预测了大麦α-淀粉酶同工酶活性位点底物结合裂中的10个结合亚位点。本文描述的大麦α-淀粉酶同工酶1（AMY 1）寡糖复合物的三维结构通过描述定义9个亚位点（即-7至+2）的残基，首次全面了解了底物结合。这些结构支持假四糖抑制剂阿卡波糖被活性酶水解。此外，还观察到糖与大麦α-淀粉酶同工酶2（AMY 2）中淀粉颗粒结合位点的结合，并比较了两种同工酶之间的糖结合模式。在AMY 1-thio-DP 4复合物中发现的“糖钳”表面结合位点在本工作中得到证实。一个网站，pupelium作为一个入口的底物的活性位点提出了在糖基部分的结合裂缝，和晶体结构的催化亲核突变体（AMY 1D 180 A）复合阿卡波糖和麦芽七糖，分别建议一个额外的作用，亲核试剂在米氏络合物的稳定。此外，可能的作用概述了表面结合位点。我们的数据支持一个模型，其中AMY 1的两个表面位点可以与直链淀粉链的自然折叠形式相互作用。由于这两个位点的特异性，它们可以定位/定向酶，以便于接近多糖链的活性位点。此外，糖钳表面位点也可以在Tyr-380的帮助下进行直链淀粉链的解开，起到“分子镊子”的作用。
Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley α-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley α-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley α-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The “sugar tongs” surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as “molecular tweezers.”