MANNOSIDASE INHIBITORS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 抑制甘露糖修剪酶人高尔基体甘露糖苷酶II（HGMII），其在N-聚糖加工途径的后期起作用，提供了阻断癌基因诱导的细胞表面寡糖结构变化的途径。 HGMII选择性切割存在于其天然底物GlcNAcMan 5GlcNAc 2中的<$（1-3）和<$（1-6）甘露糖残基。 它是一种保留糖基水解酶，采用两阶段机制，涉及位于活性位点内的两种羧酸协同作用：一种作为催化亲核试剂，另一种作为一般的酸/碱催化剂。 底物分子的环外糖基氧的质子化导致键断裂和催化亲核试剂的同时攻击以形成糖基酶中间体。 共价中间体随后被亲核水分子水解，得到构型总体保留的？-甘露糖产物。 为了探索HGMII的底物需求，我们合成了一些寡糖，用于与果蝇高尔基体甘露糖苷酶II（dGMII）的共结晶研究。 具有突变酶的共晶体结构GlcNAcMan 5，其中催化亲核酸已被Ala取代，揭示了 酶与天然底物的<$（1，2）-连接的GlcNAc部分的分子相互作用。 与从糖基结合位点到辅助GlcNAc结合位点的延伸裂缝结合的共复合物的结构与dGMII切割含有非糖基化的底物的>100倍偏好一致。 减少<$（1，2）GlcNAc残基。 相比之下，在溶酶体和动力学分析中不存在等同的GlcNAc结合位点，表明后一种酶偏好没有非还原性末端GlcNAc修饰的寡甘露糖底物，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 A co-crystal structure GlcNAcMan5 with a mutant enzyme in which the catalytic nucleophilic acid has been replaced by Ala, uncovered the molecular interactions of the enzyme with the ¿(1,2)-linked GlcNAc moiety of the natural substrate.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.