Laboratório de Planejamento e Desenvolvimento de Fármacos, Instituto de Ciências Exatas e Naturais, Universidade Federal do Pará, Belém, PA, Brazil.
J Phys Chem B. 2011 May 26;115(20):6764-75. doi: 10.1021/jp202079e. Epub 2011 May 4.
O-Glycoprotein 2-acetamino-2-deoxy-β-D-glucopyranosidase (O-GlcNAcase) hydrolyzes O-linked 2-acetamido-2-deoxy-β-D-glucopyranoside (O-GlcNAc) residues from post-translationally modified serine/threonine residues of nucleocytoplasmic protein. The chemical process involves substrate-assisted catalysis, where two aspartate residues have been identified as the two key catalytic residues of O-GlcNAcase. In this report, the first step of the catalytic mechanism used by O-GlcNAcase involving substrate-assisted catalysis has been studied using a hybrid quantum mechanical/molecular mechanical (QM/MM) Molecular Dynamics (MD) calculations. The free energy profile shows that the formation of the oxazoline intermediate in the O-GlcNAcase catalytic reaction takes place by means of a stepwise mechanism. The first step would be a cyclization of the acetomide group, which seems to be dependent on the proton transfer from a conserved aspartate, Asp298 in Clostridium perfringens O-GlcNAcase. From this new intermediate, a proton is transferred from the azoline ring to another conserved aspartate (Asp297) thus forming the oxazoline ion and departure of the aglycone. In addition, averaged values of protein-substrate interaction energy along the reaction path shows that, in fact, the transition states present the highest binding affinities. A deeper analysis of the binding contribution of the individual residues shows that Asp297, Asp298, and Asp401 are basically responsible of the stabilization of these complexes. These results would explain why O-(2-acetamido-2deoxy-d-glucopyranosylidene)amino-N-phenycarbamate (PUGNAc), 1,2-dideoxy-2'-methyl-α-D-glucopyranoso-[2,1-d]-Δ2'-thiazoline (NAG-thiazoline), and GlcNAcstatin derivatives are potent inhibitors of this enzyme, resembling the two transition states of the O-GlcNAcase catalytic reaction path. These results may be useful to rational design compounds with more interesting inhibitory activity.
O-糖蛋白 2-乙酰氨基-2-脱氧-β-D-吡喃葡萄糖苷酶（O-GlcNAcase）从核细胞质蛋白的翻译后修饰丝氨酸/苏氨酸残基上水解 O-连接的 2-乙酰氨基-2-脱氧-β-D-吡喃葡萄糖苷（O-GlcNAc）残基。该化学过程涉及底物辅助催化，其中已鉴定出两个天冬氨酸残基作为 O-GlcNAcase 的两个关键催化残基。在本报告中，使用混合量子力学/分子力学（QM/MM）分子动力学（MD）计算研究了 O-GlcNAcase 涉及底物辅助催化的催化机制的第一步。自由能曲线表明，O-GlcNAcase 催化反应中恶唑啉中间体的形成是通过逐步机制发生的。第一步将是乙酰胺基的环化，这似乎依赖于来自梭菌 O-GlcNAcase 中保守天冬氨酸的质子转移。从这个新的中间体中，质子从恶唑啉环转移到另一个保守的天冬氨酸（Asp297），从而形成恶唑啉离子并离开糖苷配基。此外，沿反应路径的蛋白质-底物相互作用能的平均值表明，实际上，过渡态具有最高的结合亲和力。对单个残基结合贡献的更深入分析表明，Asp297、Asp298 和 Asp401 基本上负责这些复合物的稳定。这些结果可以解释为什么 O-(2-乙酰氨基-2-脱氧-d-吡喃葡萄糖基)-N-苯甲酰胺基（PUGNAc）、1,2-二脱氧-2'-甲基-α-D-吡喃葡萄糖-[2,1-d]-Δ2'-噻唑啉（NAG-噻唑啉）和 GlcNAcstatin 衍生物是这种酶的有效抑制剂，类似于 O-GlcNAcase 催化反应途径的两个过渡态。这些结果可能有助于合理设计具有更有趣抑制活性的化合物。
