Manchester Interdisciplinary Biocentre and the School of Chemistry, University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
J Am Chem Soc. 2012 Aug 8;134(31):13010-7. doi: 10.1021/ja302506t. Epub 2012 Jul 30.
A fluoro-tagged N-acetylglucosamine-capped glycolipid that can form lipid microdomains in fluid phospholipid bilayers has been shown to be enzymatically galactosylated by bovine β(1,4)-galactosyltransferase. MALDI MS, HPLC, and LC-MS revealed that the rate of enzymatic transformation was significantly enhanced by lipid clustering; at a 1% mol/mol loading, clustered glycolipids were galactosylated 9-fold faster than glycolipids dispersed across the bilayer surface. The transformation of the GlcNAc "glycocalyx" into a Gal(β1-4)GlcNAc "glycocalyx" relabeled these vesicles, making them susceptible to agglutination by Erythrina cristagalli lectin (ECL). The kinetic parameters for this transformation revealed a lower apparent Km when the substrate lipids were clustered, which is attributed to multivalent binding to an extended substrate cleft around the active site. These observations may have important implications where soluble enzymes act on substrates embedded within cellular lipid rafts.
已证明，一种带有氟标记的 N-乙酰氨基葡萄糖封端的糖脂可在流体磷脂双层中形成脂质微区，能够被牛β(1,4)-半乳糖基转移酶酶促半乳糖化。MALDI MS、HPLC 和 LC-MS 表明，脂质聚集显著增强了酶转化的速率；在 1%摩尔/摩尔的负载下，聚集的糖脂的半乳糖化速度比分散在双层表面的糖脂快 9 倍。GlcNAc“糖萼”转化为 Gal(β1-4)GlcNAc“糖萼”重新标记了这些囊泡，使其容易被巴西苏木凝集素（Erythrina cristagalli lectin，ECL）凝集。这种转化的动力学参数显示，当底物脂质聚集时，表观 Km 值较低，这归因于在活性位点周围的扩展底物裂缝中多价结合。这些观察结果可能对可溶性酶在细胞脂筏内嵌入的底物上发挥作用的情况具有重要意义。
