Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase
Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase
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DOI：
10.1038/nchembio.628
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发表时间：
2011-09-01
影响因子：
14.8
通讯作者：
Davis, Benjamin G.
中科院分区：
文献类型：
--
作者：
Lee, Seung Seo;Hong, Sung You;Davis, Benjamin G.
先前确定的海藻糖-6-磷酸合成酶三元配合物的晶体结构确定了基于活性位点的有效性羟甲胺A 6'- o -磷酸和尿苷二磷酸的推定过渡态排列。线性自由能关系证实了这些抑制剂是协同过渡态模拟物，支持前向亲核攻击，涉及离去基和亲核试剂之间的氢键。动力学同位素效应表明其为高度解离的类氧碳离子过渡态。离去基团O-18效应确定了同位素敏感的键裂解，并支持亲核试剂和离去基团之间存在氢键。亲核试剂的Bronsted分析和Taft分析强调亲核试剂参与过渡态，也符合正面机制。总之，这些全面、定量的数据证实了这种不寻常的酶促反应机制。它的发现将促使人们重新评估许多生物催化剂及其底物和抑制剂。
A previously determined crystal structure of the ternary complex of trehalose-6-phosphate synthase identified a putative transition state-like arrangement based on validoxylamine A 6'-O-phosphate and uridine diphosphate in the active site. Here linear free energy relationships confirm that these inhibitors are synergistic transition state mimics, supporting front-face nucleophilic attack involving hydrogen bonding between leaving group and nucleophile. Kinetic isotope effects indicate a highly dissociative oxocarbenium ion-like transition state. Leaving group O-18 effects identified isotopically sensitive bond cleavages and support the existence of a hydrogen bond between the nucleophile and departing group. Bronsted analysis of nucleophiles and Taft analysis highlight participation of the nucleophile in the transition state, also consistent with a front-face mechanism. Together, these comprehensive, quantitative data substantiate this unusual enzymatic reaction mechanism. Its discovery should prompt useful reassessment of many biocatalysts and their substrates and inhibitors.