Coordination of manganous ion at the active site of pyruvate, phosphate dikinase: the complex of oxalate with the phosphorylated enzyme.

Electron paramagnetic resonance spectroscopy has been used to investigate the structure of the complex of manganous ion with the phosphorylated form of pyruvate,phosphate dikinase (Ep) and the inhibitor oxalate. Oxalate, an analogue of the enolate of pyruvate, is competitive with respect to pyruvate in binding to the phosphorylated form of the enzyme [Michaels, G., Milner, Y., & Reed, G.H. (1975) Biochemistry 14, 3213-3219]. Superhyperfine coupling between the unpaired electrons of Mn(II) and ligands specifically labeled with 17O has been used to identify oxygen ligands to Mn(II) in the complex with oxalate and the phosphorylated form of the enzyme. Oxalate binds at the active site as a bidentate chelate with Mn(II). An oxygen from the 3'-N-phosphohistidyl residue of the protein is in the coordination sphere of Mn(II), and at least two water molecules are also bound to Mn(II) in the complex. Oxalate also binds directly to Mn(II) in a complex with nonphosphorylated enzyme. The structure for the Ep-Mn(II)-oxalate complex implies that simultaneous coordination of a phospho group and of the attacking nucleophile to the divalent cation is likely an important factor in catalysis of this phospho-transfer reaction.