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首页> 外文期刊>Biochemistry >Discovery of Different Types of Inhibition between the Human and Thermotoga maritima alpha-Fucosidases by Fuconojirimycin-Based Derivatives
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Discovery of Different Types of Inhibition between the Human and Thermotoga maritima alpha-Fucosidases by Fuconojirimycin-Based Derivatives

机译：基于夫孔曲霉素的衍生物发现人与马氏嗜热菌α-岩藻糖苷酶之间不同类型的抑制作用

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摘要

An efficient method for examining the selectivity of inhibitors on two a-fucosidases,one from Thermotoga maritima and the other from human,was established.The X-ray crystal structure of the former enzyme makes possible the homology modeling of the human a-fucosidase,indicating the major difference between both enzymes in the periphery of the catalytic site.To investigate the difference at the molecular level,a variety of fuconojirimycin (FNJ) derivatives with substitution at C1,C2,C6,or N were rapidly prepared in microplates and screened without purification for the inhibition activities of the two alpha-fucosidases.Among the molecules that were tested,only the substitution at C1 can significantly enhance the inhibitory potency,in contrast to the control (no substitution) and compounds with substitution at other positions.The majority of Cl-substituted FNJs were found to be slow tight-binding inhibitors of the Thermotoga enzyme,while acting as the reversible inhibitors of the human fucosidase.The best inhibitor exhibited 13700-fold difference in affinity between the two enzymes,which was attributed to the dissimilar aglycon binding site.Further investigations were carried out,including site-directed mutagenesis,the comparison of K_i values among the wild type and mutants,and the intrinsic fluorescence change upon inhibitor titration,all supporting the idea that Tyr64 and Tyr267 of the Thermotoga alpha-fucosidase are critically involved in closely interacting with the aglycon of inhibitors.The increased level of contact thus induced conformational change,leading to the observed slow tight-binding inhibition.
机译：建立了一种检测抑制剂对两种α-岩藻糖苷酶的选择性的有效方法，一种酶来自海栖嗜热菌，另一种人岩藻糖酶。前一种酶的X射线晶体结构使人α-岩藻糖苷酶的同源性建模成为可能，为了研究分子水平上的差异，在微孔板中快速制备了多种在C1，C2，C6或N处被取代的岩藻霉素（FNJ）衍生物并进行了筛选。无需纯化即可纯化两个α-岩藻糖苷酶的抑制活性。在所测试的分子中，与对照品（无取代）和在其他位置具有取代基的化合物相比，仅在C1处的取代才能显着提高抑制效能。发现大多数Cl-取代的FNJs是Thermotoga酶的慢速紧密结合抑制剂，同时又是人类fuc的可逆抑制剂。最好的抑制剂表现出两种酶之间的亲和力差异是13700倍，这归因于不同的糖苷配基结合位点。进一步研究包括定点诱变，野生型和突变体之间K_i值的比较，以及抑制剂滴定后固有的荧光变化，所有这些都支持Thermotogaα-岩藻糖苷酶的Tyr64和Tyr267都与抑制剂糖苷配基紧密相互作用至关重要。接触水平的升高由此引起构象变化，从而导致观察到缓慢的紧密结合抑制。

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