The structure of the MnIIADP-nitrate-lyxose complex at the active site of hexokinase.
The coordination scheme of Mn2+ in the hexokinase-MnIIADP-nitrate-lyxose complex has been determined by electron paramagnetic resonance (EPR) spectroscopy with 17O-enriched ligands. Nitrate binds to the active site of hexokinase when MnIIADP and a sugar substrate or analogue are present. The binding of nitrate enhances inhibition by glucose when ADP is present and narrows the EPR signals of the enzyme-bound MnIIADP complex in the presence of sugar substrates or analogues. Experiments using regiospecifically 17O-enriched ADP, 17O-enriched nitrate, and 17O-enriched water establish the coordination scheme of Mn2+. The EPR experiments show that ADP is a beta-monodentate ligand and that nitrate binds directly to Mn2+. Four water molecules complete the coordination sphere of the enzyme-bound Mn2+. The dissociation constant (Kd approximately 8 mM) of nitrate for the complex with enzyme, MnIIADP, and lyxose was obtained from titration experiments. These results suggest that nitrate-stabilized, dead-end complexes of hexokinase may be useful in stabilizing the closed conformation of this "hinge-bending" enzyme for crystallographic experiments.
Institute for Enzyme Research, Graduate School, University of Wisconsin, Madison 53705.
L R
LR
Olsen
G H
GH
Reed
D000244:Adenosine Diphosphate / Q000378:metabolism*; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005656:Fungal Proteins / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism*; D008024:Ligands; D046911:Macromolecular Substances; D008345:Manganese / Q000378:metabolism*; D015394:Molecular Structure; D009566:Nitrates / Q000378:metabolism*; D010429:Pentoses / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D005656:Fungal Proteins; D008024:Ligands; D046911:Macromolecular Substances; D009566:Nitrates; D010429:Pentoses; D008345:Manganese; D000244:Adenosine Diphosphate; C043333:lyxose; D006593:Hexokinase