MANNOSIDASE INHIBITORS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 抑制甘露糖修剪酶人高尔基甘露糖苷酶II(HGMII)提供了一条阻断癌基因诱导的细胞表面寡糖结构变化的途径，HGMII在N-糖链加工途径中作用较晚。HGMII选择性地切割其天然底物GlcNAcMan5GlcNAc2中存在的(1-3)和(1-6)甘露糖基残基。它是一种保留糖基水解酶，它采用两个阶段的机制，包括位于活性中心的两个羧酸协同作用：一个作为催化亲核剂，另一个作为一般的酸/碱催化剂。底物分子的外环糖基氧的质子化导致键断裂和催化亲核试剂同时攻击以形成糖基酶中间体。随后由亲核水分子对共价中间体进行水解，得到完全保持构型的甘露糖产物。 为了探索HGMII的底物需求，我们合成了一些寡糖，并将其用于与果蝇高尔基甘露糖苷酶II(DGMII)的共结晶研究。一种共晶结构的GlcNAcMan5与突变酶，其中催化的亲核酸已被丙氨酸取代，揭示了 该酶与天然底物的β(1，2)连接的GlcNAc部分的分子相互作用。结合到从糖酮结合部位到辅助GlcNAc结合部位的延伸裂隙的共络合物的结构与dGMII对含有非-GGMII的底物的100倍切割偏好是一致的 减少(1，2)GlcNAc残基。相反，溶酶体中缺乏相应的GlcNAc结合位点，动力学分析表明，后者更喜欢没有非还原末端GlcNAc修饰的寡核苷酸底物，这表明GMII的选择性抑制剂可以利用GMII的额外结合特异性 GlcNAc结合位点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Inhibition of the mannose trimming enzyme human Golgi ¿-mannosidase II (HGMII), which acts late in the N-glycan processing pathway, provides a route to blocking the oncogene-induced changes in cell surface oligosaccharide structures. HGMII selectively cleaves ¿(1-3) and ¿(1-6) mannosyl residues present in its natural substrate GlcNAcMan5GlcNAc2. It is a retaining glycosylhydrolase, which employs a two-stage mechanism involving two carboxylic acids positioned within the active site to act in concert: one as a catalytic nucleophile and the other as a general acid/base catalyst. Protonation of the exocyclic glycosyl oxygen of a substrate molecule leads to bond-breaking and simultaneous attack of the catalytic nucleophile to form a glycosyl enzyme intermediate. Subsequent hydrolysis of the covalent intermediate by a nucleophilic water molecule gives an ¿-mannose product with overall retention of configuration. In order to probe the substrate requirements of HGMII, we have synthesized a number of oligosaccharides, which were used in co-crystallization studies with Drosophilia Golgi ¿-mannosidase II (dGMII). A co-crystal structure GlcNAcMan5 with a mutant enzyme in which the catalytic nucleophilic acid has been replaced by Ala, uncovered the molecular interactions of the enzyme with the ¿(1,2)-linked GlcNAc moiety of the natural substrate. The structure of the co-complex bound to an extended cleft leading from the glycone binding site to the accessory GlcNAc binding site is consistent with the >100-fold preference of dGMII for cleavage of substrates containing a non- reducing ¿(1,2)GlcNAc residue. By contrast, the absence of an equivalent GlcNAc binding site in lysosomal and kinetic analysis indicating that the latter enzyme prefers oligomannose substrates without non-reducing terminal GlcNAc modifications, suggests that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.