MANNOSIDASE INHIBITORS

甘露糖苷酶抑制剂

• 批准号: 8168861
• 负责人: Geert-Jan Boons
• 金额: $ 0.17万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-10 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168861
• 关键词: Acids; Active Sites; Binding; Binding Sites; Carboxylic Acids; Cell surface; Cleaved cell; Complex; Computer Retrieval of Information on Scientific Projects Database; Crystallization; Enzymes; Funding; Golgi Apparatus; Grant; Human; Hydrolysis; Institution; Kinetics; Link; Mannose; Mannosidase; Modification; Oligosaccharides; Oncogenes; Oxygen; Pathway interactions; Polysaccharides; Positioning Attribute; Process; Research; Research Personnel; Resources; Route; Source; Specificity; Staging; Structure; United States National Institutes of Health; Water; base; catalyst; inhibitor/antagonist; mutant; preference; protonation

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 A co-crystal structure GlcNAcMan5 with a mutant enzyme in which the catalytic nucleophilic acid has been replaced by Ala, uncovered the molecular interactions of the enzyme with the ¿(1,2)-linked GlcNAc moiety of the natural substrate.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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 在N-聚糖加工途径后期起作用的甘露糖修剪酶的人类高尔基酶II（HGMII）提供了阻断致癌基因诱导的细胞表面寡糖寡糖结构变化的途径。 HGMII有选择地切割（1-3）和€（1-6）甘露糖基保留其自然底物Glcnacman5GlCNAC2。它是一种固定糖基水解酶，它采用了一种两阶段的机制，涉及两个位于活性部位内的羧酸，以协同作用：一种作为催化核氧，另一个作为一般的酸/碱催化剂。底物分子的外生糖基氧的质子化会导致催化核定剂的键断裂和简单攻击形成糖基酶中间体。随后通过核视水分子对共价中间体的水解产生了``甘露糖产物'中，并具有构型的总体保留。 为了探测HGMII的底物需求，我们合成了许多寡糖，这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。一种带有突变酶的共晶结构GlcNACMAN5，其中催化核苯甲酸已被ALA取代，发现了酶与自然底物的链接GlcNAC的分子相互作用。与辅助GlcNAC结合位点导致延伸到延伸的裂缝结合的共复合物的结构与DGMII的> 100倍偏爱对于裂解含有非还原的GLCNAC居住的底物的裂解。相比之下，在溶酶体和动力学分析中缺乏等效的GlcNAC结合位点表明，后一种酶优先于寡头糖底物而没有非还原末端GLCNAC修饰，这表明对GMII的选择性抑制剂的选择性抑制剂可以利用GLCNAC结合的其他结合特异性。