Zechel David L, Boraston Alisdair B, Gloster Tracey, Boraston Catherine M, Macdonald James M, Tilbrook D Matthew G, Stick Robert V, Davies Gideon J
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, United Kingdom.
J Am Chem Soc. 2003 Nov 26;125(47):14313-23. doi: 10.1021/ja036833h.
The design and synthesis of transition-state mimics reflects the growing need both to understand enzymatic catalysis and to influence strategies for therapeutic intervention. Iminosugars are among the most potent inhibitors of glycosidases. Here, the binding of 1-deoxynojirimycin and (+)-isofagomine to the "family GH-1" beta-glucosidase of Thermotoga maritima is investigated by kinetic analysis, isothermal titration calorimetry, and X-ray crystallography. The binding of both of these iminosugar inhibitors is driven by a large and favorable enthalpy. The greater inhibitory power of isofagomine, relative to 1-deoxynojirimycin, however, resides in its significantly more favorable entropy; indeed the differing thermodynamic signatures of these inhibitors are further highlighted by the markedly different heat capacity values for binding. The pH dependence of catalysis and of inhibition suggests that the inhibitory species are protonated inhibitors bound to enzymes whose acid/base and nucleophile are ionized, while calorimetry indicates that one proton is released from the enzyme upon binding at the pH optimum of catalysis (pH 5.8). Given that these results contradict earlier proposals that the binding of racemic isofagomine to sweet almond beta-glucosidase was entropically driven (Bülow, A. et al. J. Am. Chem. Soc. 2000, 122, 8567-8568), we reinvestigated the binding of 1-deoxynojirimycin and isofagomine to the sweet almond enzyme. Calorimetry confirms that the binding of isofagomine to sweet almond beta-glucosidases is, as observed for the T. maritima enzyme, driven by a large favorable enthalpy. The crystallographic structures of the native T. maritima beta-glucosidase, and its complexes with isofagomine and 1-deoxynojirimycin, all at approximately 2.1 A resolution, reveal that additional ordering of bound solvent may present an entropic penalty to 1-deoxynojirimycin binding that does not penalize isofagomine.
过渡态模拟物的设计与合成反映出人们对于理解酶催化作用以及影响治疗干预策略的需求日益增长。亚氨基糖是糖苷酶最有效的抑制剂之一。在此，通过动力学分析、等温滴定量热法和X射线晶体学研究了1-脱氧野尻霉素和(+)-异法戈明与嗜热栖热菌“GH-1家族”β-葡萄糖苷酶的结合情况。这两种亚氨基糖抑制剂的结合均由较大的有利焓驱动。然而，相对于1-脱氧野尻霉素，异法戈明更强的抑制能力在于其显著更有利的熵；实际上，这些抑制剂不同的热力学特征通过结合时明显不同的热容值得到了进一步凸显。催化和抑制的pH依赖性表明，抑制性物种是与酸/碱和亲核试剂已电离的酶结合的质子化抑制剂，而量热法表明在催化的最适pH（pH 5.8）下结合时，酶会释放出一个质子。鉴于这些结果与早期关于外消旋异法戈明与甜杏仁β-葡萄糖苷酶的结合是由熵驱动的提议相矛盾（比洛，A.等人，《美国化学会志》，2000年，122卷，8567 - 8568页），我们重新研究了1-脱氧野尻霉素和异法戈明与甜杏仁酶的结合。量热法证实，如同在嗜热栖热菌酶中观察到的那样，异法戈明与甜杏仁β-葡萄糖苷酶的结合是由较大的有利焓驱动的。嗜热栖热菌天然β-葡萄糖苷酶及其与异法戈明和1-脱氧野尻霉素的复合物的晶体结构，分辨率均约为2.1埃，结果表明，结合溶剂的额外有序排列可能会给1-脱氧野尻霉素的结合带来熵罚，而异法戈明则不受此影响。
