Carrell H L, Glusker J P, Burger V, Manfre F, Tritsch D, Biellmann J F
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111.
Proc Natl Acad Sci U S A. 1989 Jun;86(12):4440-4. doi: 10.1073/pnas.86.12.4440.
The structures of crystalline D-xylose isomerase (D-xylose ketol-isomerase; EC 5.3.1.5) from Streptomyces rubiginosus and of its complexes with substrate and with an active-site-directed inhibitor have been determined by x-ray diffraction techniques and refined to 1.9-A resolution. This study identifies the active site, as well as two metal-binding sites. The metal ions are important in maintaining the structure of the active-site region and one of them binds C3-O and C5-O of the substrate forming a six-membered ring. This study has revealed a very close contact between histidine and C1 of a substrate, suggesting that this is the active-site base that abstracts a proton from substrate. The mechanism-based inhibitor is a substrate analog and is turned over by the enzyme to give a product that alkylates this same histidine, reinforcing our interpretation. The changes in structure of the native enzyme, the enzyme with bound substrate, and the alkylated enzyme indicate that the mechanism involves an "open-chain" conformation of substrate and that the intermediate in the isomerization reaction is probably a cis-ene diol because the active-site histidine is correctly placed to abstract a proton from C1 or C2 of the substrate. A water molecule binds to C1O and C2O of the substrate and so may act as a proton donor or acceptor in the enolization of a ring-opened substrate.
通过X射线衍射技术测定了来自红色链霉菌的结晶D-木糖异构酶（D-木糖酮醇异构酶；EC 5.3.1.5）及其与底物和活性位点导向抑制剂的复合物的结构，并将其精修至1.9埃分辨率。这项研究确定了活性位点以及两个金属结合位点。金属离子对于维持活性位点区域的结构很重要，其中一个金属离子与底物的C3-O和C5-O结合形成一个六元环。这项研究揭示了组氨酸与底物的C1之间有非常紧密的接触，表明这就是从底物夺取质子的活性位点碱基。基于机制的抑制剂是一种底物类似物，被酶转化为一种产物，该产物使同一个组氨酸烷基化，这加强了我们 的解释。天然酶、结合底物的酶和烷基化酶的结构变化表明，该机制涉及底物的“开链”构象，异构化反应中的中间体可能是顺式烯二醇，因为活性位点组氨酸的位置正确，可以从底物的C1或C2夺取质子。一个水分子与底物的C1O和C2O结合，因此可能在开环底物的烯醇化过程中作为质子供体或受体。
