Probing Synergy between Two Catalytic Strategies in the Glycoside Hydrolase O-GlcNAcase Using Multiple Linear Free Energy Relationships
Probing Synergy between Two Catalytic Strategies in the Glycoside Hydrolase O-GlcNAcase Using Multiple Linear Free Energy Relationships
复制标题
DOI：
10.1021/ja904506u
复制
发表时间：
2009-09-23
影响因子：
15
通讯作者：
Vocadlo, David J.
中科院分区：
文献类型：
--
作者：
Greig, Ian R.;Macauley, Matthew S.;Vocadlo, David J.
人O-GlcNAc酶在调节丝氨酸和苏氨酸残基与β-O-连接的N-乙酰葡萄糖胺单糖单元（O-GlcNAc）的翻译后修饰中起重要作用。O-GlcNAcase的机制涉及底物的2-乙酰氨基的亲核参与以置换糖苷连接的离去基团。这种酶对底物结构变化的耐受性使我们能够使用几个系列的底物来表征O-GlcNAcase过渡态，以产生多个同时的自由能关系。模式揭示的变化机制，过渡态，和速率决定步骤后，同时变化的亲核强度和离去基团的能力进行了观察。所观察到的机理变化反映了酶的一般酸和催化亲核试剂所起的作用。值得注意的是，这些结果说明了酶如何协同利用两种催化模式;这是许多小分子催化模型无法实现的特征。这些研究还表明，动力学意义的oxocarbenium离子中间体的O-GlcNAc酶催化水解的氨基葡萄糖苷，探测的限制，可以学到什么使用nonatomistic，酶的过渡态结构的调查，并提供一般的见解如何保留糖苷水解酶的超家族作为有效的催化剂。
Human O-GlcNAcase plays an important role in regulating the post-translational modification of serine and threonine residues with beta-O-Linked N-acetylglucosamine monosaccharide unit (O-GlcNAc). The mechanism of O-GlcNAcase involves nucleophilic participation of the 2-acetamido group of the substrate to displace a glycosidically linked leaving group. The tolerance of this enzyme for variation in substrate structure has enabled us to characterize O-GlcNAcase transition states using several series of substrates to generate multiple simultaneous free-energy relationships. Patterns revealing changes in mechanism, transition state, and rate-determining step upon concomitant variation of both nucleophilic strength and leaving group abilities are observed. The observed changes in mechanism reflect the roles played by the enzymic general acid and the catalytic nucleophile. Significantly, these results illustrate how the enzyme synergistically harnesses both modes of Catalysis; a feature that eludes many small molecule models of catalysis. These studies also suggest the kinetic significance of an oxocarbenium ion intermediate in the O-GlcNAcase-catalyzed hydrolysis of glucosaminides, probing the limits of what may be learned using nonatomistic, investigations of enzymic transition-state structure and offering general insights into how the superfamily of retaining glycoside hydrolases act as efficient catalysts.