Characterization of ligand binding to N-acetylglucosamine kinase studied by STD NMR.
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.
Blume
Astrid
A
Institut für Chemie, Universität zu Lübeck, Germany. asblume@gmx.net
Berger
Markus
M
Benie
Andrew J
AJ
Peters
Thomas
T
Hinderlich
Stephan
S
D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D019281:Dimerization; D006801:Humans; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D015394:Molecular Structure; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D012996:Solutions / Q000737:chemistry; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry
D008024:Ligands; D012996:Solutions; D014867:Water; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C018716:N-acetylglucosamine kinase; D008274:Magnesium; D000117:Acetylglucosamine