Blume Astrid, Berger Markus, Benie Andrew J, Peters Thomas, Hinderlich Stephan
Institut für Chemie, Universität zu Lübeck, Germany.
Biochemistry. 2008 Dec 9;47(49):13138-46. doi: 10.1021/bi8016894.
Saturation transfer difference (STD) NMR experiments on human N-acetylglucosamine kinase (GlcNAc kinase) have been used to determine binding epitopes for the GlcNAc and ATP substrates and their analogues. The study reveals that during the enzyme reaction the binding mode of both substrates is conserved, although the binding affinity of the sugar is reduced. This suggests that the protein does not undergo any significant structural changes during catalysis. Our experiments also demonstrate that GlcNAc kinase has residual activity in the absence of Mg(2+). Furthermore, our experiments clearly show that the GlcNAc kinase predominately, if not exclusively, produces the beta anomer of phosphorylated sugars. To identify the minimum requirements for substrate binding, a detailed analysis of different natural occurring as well as synthetic sugars was employed. Modifications at the 1, 2, 3, 4 and 6 position as well as the N-acetyl group greatly reduce the binding affinity. In addition, the binding mode of these substrate analogues is often also changed. The high beta anomeric preference of GlcNAc kinase along with the drastically reduced binding affinity for sugars other than GlcNAc, suggests that GlcNAc kinase phosphorylates beta-GlcNAc in cells.
利用饱和转移差（STD）核磁共振实验对人N-乙酰葡糖胺激酶（GlcNAc激酶）进行研究，以确定GlcNAc和ATP底物及其类似物的结合表位。该研究表明，在酶反应过程中，尽管糖的结合亲和力降低，但两种底物的结合模式是保守的。这表明蛋白质在催化过程中不会发生任何显著的结构变化。我们的实验还证明，在没有Mg(2+)的情况下，GlcNAc激酶仍具有残余活性。此外，我们的实验清楚地表明，GlcNAc激酶主要（如果不是唯一地）产生磷酸化糖的β异头物。为了确定底物结合的最低要求，我们对不同的天然存在的以及合成的糖进行了详细分析。在1、2、3、4和6位以及N-乙酰基上的修饰大大降低了结合亲和力。此外，这些底物类似物的结合模式通常也会发生变化。GlcNAc激酶对β异头物的高度偏好以及对除GlcNAc以外的糖的结合亲和力急剧降低，表明GlcNAc激酶在细胞中使β-GlcNAc磷酸化。
