O-GlcNAcase uses substrate-assisted catalysis -: Kinetic analysis and development of highly selective mechanism-inspired inhibitors
O-GlcNAcase uses substrate-assisted catalysis -: Kinetic analysis and development of highly selective mechanism-inspired inhibitors
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DOI：
10.1074/jbc.m413819200
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发表时间：
2005-07-08
影响因子：
4.8
通讯作者：
Vocadlo, DJ
中科院分区：
文献类型：
--
作者：
Macauley, MS;Whitworth, GE;Vocadlo, DJ
2-乙酰氨基-2-脱氧-D-吡喃葡萄糖（GlcNAc）对核质蛋白的丝氨酸和苏氨酸残基的翻译后修饰是涉及多个细胞过程的可逆过程。酶O-GlcNAc酶催化β-O-连接的GlcNAc（O-GlcNAc）从修饰的蛋白质中裂解，是84糖苷水解酶家族的成员。家族20 β-氨基己糖苷酶没有明显的序列相似性，但在功能上与O-GlcNAc酶相关，因为两种酶都从糖缀合物上切割末端GlcNAc残基。已知溶酶体β-氨基己糖苷酶使用涉及底物的2-乙酰氨基的底物辅助催化;然而，人O-GlcNAcase的催化机制尚不清楚。通过使用一系列4-甲基伞形酮基2-脱氧-2-N-氟乙酰基-β-D-吡喃葡萄糖苷底物，这些酶的Taft样线性自由能分析表明O-GlcNAcase使用涉及邻位辅助的催化机制。与该提议一致，1，2-双脱氧-2 '-甲基-α-D-吡喃葡萄糖基-[2，1-d]-Delta 2'-噻唑啉是一种模拟20家族糖苷水解酶催化机制中提出的恶唑啉中间体的抑制剂，已被证明是O-GlcNAcase（K-I = 0.070 μ M）和β-氨基己糖苷酶（KI = 0.070 μ M）的有效竞争性抑制剂。制备了一系列1，2-二脱氧-2 ′-甲基-α-D-吡喃葡萄糖[2，1-d]-δ 2 ′-噻唑啉类似物，一种抑制剂对O-GlcNAcase（K-I = 0.230 μ M）的选择性是对β-氨基己糖苷酶（K-I = 340 μ M）的1500倍。这些抑制剂是细胞可渗透的，并调节组织培养物中O-GlcNAcase的活性。由于这两种酶在生物体健康中具有重要作用，因此O-GlcNAcase的这些有效和选择性抑制剂应证明可用于研究该酶在生物体水平上的作用，而不会产生源于β-氨基己糖苷酶的伴随抑制的复杂化学表型。
The post-translational modification of serine and threonine residues of nucleocytoplasmic proteins with 2-acetamido-2-deoxy-D-glucopyranose ( GlcNAc) is a reversible process implicated in multiple cellular processes. The enzyme O-GlcNAcase catalyzes the cleavage of beta-O-linked GlcNAc (O-GlcNAc) from modified proteins and is a member of the family 84 glycoside hydrolases. The family 20 beta-hexosaminidases bear no apparent sequence similarity yet are functionally related to O-GlcNAcase because both enzymes cleave terminal GlcNAc residues from glycoconjugates. Lysosomal beta-hexosaminidase is known to use substrate-assisted catalysis involving the 2-acetamido group of the substrate; however, the catalytic mechanism of human O-GlcNAcase is unknown. By using a series of 4-methylumbelliferyl 2-deoxy-2-N-fluoroacetyl-beta-D-glucopyranoside substrates, Taft-like linear free energy analyses of these enzymes indicates that O-GlcNAcase uses a catalytic mechanism involving anchimeric assistance. Consistent with this proposal, 1,2-dideoxy-2'-methyl-alpha-D-glucopyranoso-[2,1-d]-Delta 2'-thiazoline, an inhibitor that mimics the oxazoline intermediate proposed in the catalytic mechanism of family 20 glycoside hydrolases, is shown to act as a potent competitive inhibitor of both O-GlcNAcase (K-I = 0.070 mu M) and beta-hexosaminidase (KI = 0.070 mu M). A series of 1,2-dideoxy-2'-methyl-alpha-D-glucopyranoso[2,1-d]-Delta 2'-thiazoline analogues were prepared, and one inhibitor demonstrated a remarkable 1500-fold selectivity for O-GlcNAcase (K-I = 0.230 mu M) over beta-hexosaminidase (K-I = 340 mu M). These inhibitors are cell permeable and modulate the activity of O-GlcNAcase in tissue culture. Because both enzymes have vital roles in organismal health, these potent and selective inhibitors of O-GlcNAcase should prove useful in studying the role of this enzyme at the organismal level without generating a complex chemical phenotype stemming from concomitant inhibition of beta-hexosaminidase.