DESIGN, SYNTHESIS, AND CHARACTERIZATION OF A POTENT XYLOSE ISOMERASE INHIBITOR, D-THREONOHYDROXAMIC ACID, AND HIGH-RESOLUTION X-RAY CRYSTALLOGRAPHIC STRUCTURE OF THE ENZYME-INHIBITOR COMPLEX
DESIGN, SYNTHESIS, AND CHARACTERIZATION OF A POTENT XYLOSE ISOMERASE INHIBITOR, D-THREONOHYDROXAMIC ACID, AND HIGH-RESOLUTION X-RAY CRYSTALLOGRAPHIC STRUCTURE OF THE ENZYME-INHIBITOR COMPLEX
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DOI：
10.1021/bi00011a032
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发表时间：
1995-03-21
期刊：
影响因子：
2.9
通讯作者：
RINGE, D
中科院分区：
文献类型：
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作者：
ALLEN, KN;LAVIE, A;RINGE, D
结合的一个有效的抑制剂的酶D-木糖异构酶从链霉菌olivochromogenes的动力学和X-射线晶体学检查。抑制剂D-苏氨异羟肟酸（THA）的设计，以模拟假定的过渡态的异构化的酶催化底物木糖的步骤。THA的合成和发现是一个缓慢结合的竞争性抑制剂与底物葡萄糖。小于或等于IOOnM的Ki比葡萄糖的Ki小至少一百万倍。将THA浸泡到晶体中（浓度= 1000 K（i））的木糖异构酶的X射线晶体学结构获得1.6埃的分辨率，并精修到21.6%的R因子。游离酶和木糖异构酶-THA复合物中的酶没有显示出显著的结构差异。THA以类似的方式与葡萄糖结合，呈线性构象，与Mg-1和Mg-2形成配体，与His 53和Lys 182形成氢键。在这些相似性的基础上，葡萄糖结合和其有效的抑制，我们建议，THA类似于酶催化的氢化物转移反应的过渡态。THA C2羟基在Mg-1和Mg-2之间形成桥连配体;它必须去质子化才能实现。通过类比，我们提出，在催化反应过程中，C2的底物葡萄糖是去质子化的，这个质子可以移动到C1羟基伴随着氢化物转移。我们发现在催化过程中的C2羟基去质子化后的金属运动的证据，允许形成的桥接配体。此外，在木糖异构酶-THA复合物中观察到结合到Mg-2而不是氢氧化物的水分子（如天然结构中所见）表明羟基的质子化发生在开环之后的步骤中。与金属结合的氢氧离子可以作为通用碱使底物的C2羟基去质子化。由于它们作为金属配体的能力，异羟肟酸可以是双金属酶的一般抑制剂。
The binding of a potent inhibitor to the enzyme D-xylose isomerase from Streptomyces olivochromogenes was examined by kinetics and X-ray crystallography. The inhibitor D-threonohydroxamic acid (THA) was designed to mimic the putative transition state of the isomerization step catalyzed by the enzyme on the substrate xylose. THA was synthesized and found to be a slow-binding competitive inhibitor with the substrate glucose. The K-i less than or equal to 100 nM was at least one million-fold less than the K-M for glucose. The X-ray crystallographic structure of xylose isomerase with THA soaked into the crystals (concentration = 1000K(i)) was obtained to 1.6-Angstrom resolution and refined to an R factor of 21.6%. The free enzyme and the enzyme in the xylose isomerase-THA complex show no significant structural differences. THA binds in an analogous fashion to glucose, in a linear conformation, forming ligands with Mg-1 and Mg-2 and hydrogen bonds with His53 and Lys182. On the basis of these similarities to glucose binding and its potent inhibition, we propose that THA resembles the transition state for the enzyme-catalyzed hydride transfer reaction. The THA C2 hydroxyl forms a bridging ligand between Mg-1 and Mg-2; it must be deprotonated to do so. By analogy, we propose that, during the catalytic reaction, C2 of the substrate glucose is deprotonated, and that this proton can be moved to the C1 hydroxyl concomitant with hydride transfer. We find evidence for metal movement during catalysis upon deprotonation of the C2 hydroxyl, to allow formation of a bridging ligand. Ln addition, the observation in the xylose isomerase-THA complex of a water molecule bound to Mg-2 instead of a hydroxide (as seen in the native structure) suggests that protonation of the hydroxyl occurs in a step following ring opening. The metal-bound hydroxide ion may act as a general base to deprotonate the C2 hydroxyl of the substrate. Due to their ability to act as metal ligands, hydroxamic acids may be general inhibitors of dimetallic enzymes.