Vallée F, Kadziola A, Bourne Y, Juy M, Rodenburg K W, Svensson B, Haser R
Macromolécules Biologiques, UPR 9039, Institut de Biologie Structurale et Microbiologie, CNRS, France.
Structure. 1998 May 15;6(5):649-59. doi: 10.1016/s0969-2126(98)00066-5.
Barley alpha-amylase is a 45 kDa enzyme which is involved in starch degradation during barley seed germination. The released sugars provide the plant embryo with energy for growth. The major barley alpha-amylase isozyme (AMY2) binds with high affinity to the endogenous inhibitor BASI (barley alpha-amylase/subtilisin inhibitor) whereas the minor isozyme (AMY1) is not inhibited. BASI is a 19.6 kDa bifunctional protein that can simultaneously inhibit AMY2 and serine proteases of the subtilisin family. This inhibitor may therefore prevent degradation of the endosperm starch during premature sprouting and protect the seed from attack by pathogens secreting proteases.
The crystal structure of AMY2 in complex with BASI was determined and refined at 1.9 A resolution. BASI consists of a 12-stranded beta-barrel structure which belongs to the beta-trefoil fold family and inhibits AMY2 by sterically occluding access of the substrate to the active site of the enzyme. The AMY2-BASI complex is characterized by an unusual completely solvated calcium ion located at the protein-protein interface.
The AMY2-BASI complex represents the first reported structure of an endogenous protein-protein complex from a higher plant. The structure of the complex throws light on the strict specificity of BASI for AMY2, and shows that domain B of AMY2 contributes greatly to the specificity of enzyme-inhibitor recognition. In contrast to the three-dimensional structures of porcine pancreatic alpha-amylase in complex with proteinaceous inhibitors, the AMY2-BASI structure reveals that the catalytically essential amino acid residues of the enzyme are not directly bound to the inhibitor. Binding of BASI to AMY2 creates a cavity, exposed to the external medium, that is ideally shaped to accommodate an extra calcium ion. This feature may contribute to the inhibitory effect, as the key amino acid sidechains of the active site are in direct contact with water molecules which are in turn ligated to the calcium ion.
大麦α-淀粉酶是一种45 kDa的酶，参与大麦种子萌发过程中的淀粉降解。释放出的糖类为植物胚胎的生长提供能量。主要的大麦α-淀粉酶同工酶（AMY2）与内源性抑制剂BASI（大麦α-淀粉酶/枯草杆菌蛋白酶抑制剂）具有高亲和力结合，而次要同工酶（AMY1）则不受抑制。BASI是一种19.6 kDa的双功能蛋白，可同时抑制AMY2和枯草杆菌蛋白酶家族的丝氨酸蛋白酶。因此，这种抑制剂可能会防止胚乳淀粉在过早发芽期间降解，并保护种子免受分泌蛋白酶的病原体的侵袭。
确定了与BASI复合的AMY2的晶体结构，并在1.9 Å分辨率下进行了优化。BASI由一个12股β-桶状结构组成，属于β-三叶折叠家族，通过空间位阻阻止底物进入酶的活性位点来抑制AMY2。AMY2-BASI复合物的特征是在蛋白质-蛋白质界面处有一个异常的完全溶剂化的钙离子。
AMY2-BASI复合物代表了高等植物中首次报道的内源性蛋白质-蛋白质复合物的结构。该复合物的结构揭示了BASI对AMY2的严格特异性，并表明AMY2的结构域B对酶-抑制剂识别的特异性有很大贡献。与猪胰α-淀粉酶与蛋白质抑制剂复合的三维结构不同，AMY2-BASI结构表明该酶的催化必需氨基酸残基不直接与抑制剂结合。BASI与AMY2的结合形成了一个暴露于外部介质的腔，其形状理想地适合容纳一个额外的钙离子。这一特征可能有助于抑制作用，因为活性位点的关键氨基酸侧链直接与水分子接触，而水分子又与钙离子相连。
