Hartman Matthew C T, Jiang Songmin, Rush Jeffrey S, Waechter Charles J, Coward James K
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Biochemistry. 2007 Oct 16;46(41):11630-8. doi: 10.1021/bi700863s. Epub 2007 Sep 21.
In glycosyltransferase-catalyzed reactions a new carbohydrate-carbohydrate bond is formed between a carbohydrate acceptor and the carbohydrate moiety of either a sugar nucleotide donor or lipid-linked saccharide donor. It is currently believed that most glycosyltransferase-catalyzed reactions occur via an electrophilic activation mechanism with the formation of an oxocarbenium ion-like transition state, a hypothesis that makes clear predictions regarding the charge development on the donor (strong positive charge) and acceptor (minimal negative charge) substrates. To better understand the mechanism of these enzyme-catalyzed reactions, we have introduced a strongly electron-withdrawing group (fluorine) at C-5 of both donor and acceptor substrates in order to explore its effect on catalysis. In particular, we have investigated the effects of the 5-fluoro analogues on the kinetics of two glycosyltransferase-catalyzed reactions mediated by UDP-GlcNAc:GlcNAc-P-P-Dol N-acetylglucosaminyltransferase (chitobiosyl-P-P-lipid synthase, CLS) and beta-N-acetylglucosaminyl-beta-1,4 galactosyltransferase (GalT). The 5-fluoro group has a marked effect on catalysis when inserted into the UDP-GlcNAc donor, with the UDP(5-F)-GlcNAc serving as a competitive inhibitor of CLS rather than a substrate. The (5-F)-GlcNAc beta-octyl glycoside acceptor, however, is an excellent substrate for GalT. Both of these results support a weakly associative transition state for glycosyltransferase-catalyzed reactions that proceed with inversion of configuration.
在糖基转移酶催化的反应中，新的碳水化合物-碳水化合物键在碳水化合物受体与糖核苷酸供体或脂质连接的糖供体的碳水化合物部分之间形成。目前认为，大多数糖基转移酶催化的反应通过亲电活化机制发生，形成氧鎓离子样过渡态，这一假说对供体（强正电荷）和受体（最小负电荷）底物上电荷的发展做出了明确预测。为了更好地理解这些酶催化反应的机制，我们在供体和受体底物的C-5位引入了一个强吸电子基团（氟），以探索其对催化作用的影响。特别是，我们研究了5-氟类似物对由UDP-GlcNAc介导的两种糖基转移酶催化反应动力学的影响：UDP-GlcNAc：GlcNAc-P-P-Dol N-乙酰葡糖胺基转移酶（壳二糖基-P-P-脂质合酶，CLS）和β-N-乙酰葡糖胺基-β-1,4-半乳糖基转移酶（GalT）。当5-氟基团插入UDP-GlcNAc供体时，对催化作用有显著影响，UDP(5-F)-GlcNAc作为CLS的竞争性抑制剂而非底物。然而，(5-F)-GlcNAcβ-辛基糖苷受体是GalT的优良底物。这两个结果都支持了糖基转移酶催化反应的弱缔合过渡态，该反应以构型翻转的方式进行。
