Mikami B, Hehre E J, Sato M, Katsube Y, Hirose M, Morita Y, Sacchettini J C
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
Biochemistry. 1993 Jul 13;32(27):6836-45. doi: 10.1021/bi00078a006.
New crystallographic findings are presented which offer a deeper understanding of the structure and functioning of beta-amylase, the first known exo-type starch-hydrolyzing enzyme. A refined three-dimensional structure of soybean beta-amylase, complexed with the inhibitor alpha-cyclodextrin, has been determined at 2.0-A resolution with a conventional R-value of 17.5%. The model contains 491 amino acid residues, 319 water molecules, 1 sulfate ion, and 1 alpha-cyclodextrin molecule. The protein consists of a core with an (alpha/beta)8 supersecondary structure, plus a smaller globular region formed by long loops (L3, L4, and L5) extending from beta-strands beta 3, beta 4, and beta 5. Between the two regions is a cleft that opens into a pocket whose floor contains the postulated catalytic center near the carboxyl group of Glu 186. The annular alpha-cyclodextrin binds in (and partly projects from) the cleft with its glucosyl O-2/O-3 face abutting the (alpha/beta)8 side and with its alpha-D(1 --> 4) glucosidic linkage progression running clockwise as viewed from that side. The ligand does not bind deeply enough to interact with the carboxyl group of Glu 186. Rather, it occupies most of the cleft entrance, strongly suggesting that alpha-cyclodextrin inhibits catalysis by blocking substrate access to the more deeply located reaction center. Of the various alpha-cyclodextrin interactions with protein residues in loops L4, L5, L6, and L7, most notable is the shallow inclusion complex formed with Leu 383 (in L7, on the core side of the cleft) through contacts of its methyl groups with the C-3 atoms of four of the ligand's D-glucopyranosyl residues. All six residues of the bound alpha-cyclodextrin are of 4C1 conformation and are joined by alpha-1,4 linkages with similar torsional angles to form a nearly symmetrical torus as reported for crystalline inclusion complexes with alpha-cyclodextrin. We envision a significant role for the methyl groups of Leu 383 at the cleft entrance with respect to the productive binding of the outer chains of starch.
本文展示了新的晶体学研究结果，这些结果有助于更深入地理解β-淀粉酶的结构和功能，β-淀粉酶是已知的第一种外切型淀粉水解酶。已确定了与抑制剂α-环糊精复合的大豆β-淀粉酶的精细三维结构，分辨率为2.0埃，传统R值为17.5%。该模型包含491个氨基酸残基、319个水分子、1个硫酸根离子和1个α-环糊精分子。蛋白质由一个具有(α/β)8超二级结构的核心以及一个由从β链β3、β4和β5延伸出的长环（L3、L4和L5）形成的较小球状区域组成。在这两个区域之间是一个裂隙，通向一个口袋，口袋底部在Glu 186羧基附近包含假定的催化中心。环状α-环糊精以其葡糖基O-2/O-3面紧靠(α/β)8面的方式结合在裂隙中（并部分从裂隙中伸出），并且从该面看其α-D(1→4)糖苷键的方向为顺时针。配体结合得不够深，无法与Glu 186的羧基相互作用。相反，它占据了大部分裂隙入口，强烈表明α-环糊精通过阻止底物进入更深层的反应中心来抑制催化作用。在α-环糊精与环L4、L5、L6和L7中的蛋白质残基的各种相互作用中，最显著的是与Leu 383（在L7中，位于裂隙的核心侧）形成的浅包合复合物，其甲基与配体的四个D-吡喃葡萄糖残基的C-3原子接触。结合的α-环糊精的所有六个残基均为4C1构象，并通过具有相似扭转角的α-1,4键连接，形成一个几乎对称的环面，这与报道的α-环糊精晶体包合复合物的情况相同。我们设想位于裂隙入口的Leu 383的甲基对于淀粉外链的有效结合具有重要作用。
