Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants
Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants
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DOI：
10.1021/bi052370b
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发表时间：
2006-03-21
期刊：
影响因子：
2.9
通讯作者：
Vocadlo, DJ
中科院分区：
文献类型：
--
作者：
Çetinbas, N;Macauley, MS;Vocadlo, DJ
O-GlcNAcase 是家族 84 β-N-乙酰氨基葡萄糖苷酶，催化 β-O-连接的 2-乙酰氨基-2-脱氧-D-吡喃葡萄糖 (O-GlcNAc) 从翻译后修饰蛋白的丝氨酸和苏氨酸残基上水解裂解。 O-GlcNAcases 使用双置换机制，涉及瞬时双环恶唑啉中间体的形成和分解。然而，任何家族 84 酶促进该反应的关键催化残基尚不清楚。由于这些残基在家族 84 糖苷水解酶中高度保守，因此产生了人 O-GlcNAcase 的两个突变体 D174A 和 D175A。 D174A 突变酶的结构反应性研究揭示了多种底物的催化活性严重受损，同时 pH 活性谱与关键催化残基的删除一致。 D175A 突变酶显示，对于具有较差离去基团的底物，催化效率显着降低（高达 3000 倍），而对于具有良好先导基团的底物，差异要小得多（7 倍）。这种突变酶还可以裂解硫代糖苷，其催化效率与野生型酶基本相同。此外，添加叠氮化物作为外源亲核试剂会增加该酶对带有优异离去基团的底物的活性。总之，这些结果允许明确指定 Asp(174) 为极化 2-乙酰胺基以攻击异头中心的残基，而 Asp(175) 为充当一般酸/碱催化剂的残基。因此，家族84糖苷水解酶使用DD催化对来实现催化作用。
O-GlcNAcase is a family 84 beta-N-acetylglucosaminidase catalyzing the hydrolytic cleavage of beta-O-linked 2-acetamido-2-deoxy-D-glycopyranose (O-GlcNAc) from serine and threonine residues of posttranslationally modified proteins. O-GlcNAcases use a double-displacement mechanism involving formation and breakdown of a transient bicyclic oxazoline intermediate. The key catalytic residues of any family 84 enzyme facilitating this reaction, however, are unknown. Two mutants of human O-GlcNAcase, D174A and D175A, were generated since these residues are highly conserved among family 84 glycoside hydrolases. Structure-reactivity studies of the D174A mutant enzyme reveals severely impaired catalytic activity across a broad range of substrates alongside a pH-activity profile consistent with deletion of a key catalytic residue. The D175A mutant enzyme shows a significant decrease in catalytic efficiency with Substrates bearing poor leaving groups (up to 3000-fold), while for substates bearing good leading groups the difference is much smaller (7-fold). This mutant enzyme also cleaves thioglycosides with essentially the same catalytic efficiency as the wild-type enzyme. As well, addition of azide as an exogenous nucleophile increases the activity of this enzyme toward a substrate bearing an excellent leaving group. Together, these results allow unambiguous assignment of Asp(174) as the residue that polarizes the 2-acetamido group for attack on the anomeric center and Asp(175) as the residue that Functions as the general acid/base catalyst. Therefore, the family 84 glycoside hydrolases use a DD catalytic pair to effect catalysis.