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ABSTRACT: Saturation transfer difference (STD) NMR experiments on human N-acetylglucosamine kinase (GlcNAc kinase) have been used to determine binding epitopes for the G1cNAc 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 G1cNAc kinase has residual activity in the absence of Mg2+. Furthermore, our experiments clearly show that the GlcNAc kinase predominately, if not exclusively, produces the α 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 β anomeric preference of G1cNAc kinase along with the drastically reduced binding affinity for sugars other than G1cNAc, suggests that GlcNAc kinase phosphorylates fi-G1cNAc in cells. [ABSTRACT FROM AUTHOR]