Vanadyl(IV) complexes with pyruvate kinase: structure-activity correlations

Pyruvate kinase is unusual among kinases in that it requires two equivalents of divalent cation as cofactors, and, in addition, has an absolute requirement for monovalent cations. Vanadyl ion (V(IV)O2+) was employed as a paramagnetic probe of the metal sites. Electron paramagnetic resonance spectroscopy (EPR) provided structural details concerning the interactions of the substrates and cofactors, and the structural findings have been correlated with important aspects in catalysis. Vanadyl ion is essentially inactive in the normal reaction of pyruvate kinase (P-enolpyruvate + ADP + H+ → pyruvate + ATP), and in the enolization of pyruvate. However, significant activation is observed in the phosphorylation of the alternative substrate, glycolate, by ATP, and the vanadyl-enzyme exhibits a high HCO3-stimulated ATPase activity compared to Mg2+ or Mn2+. EPR spectra from ternary complexes with 17O-enriched oxalate, pyruvate and glycolate show inhomogeneous broadening, indicating all three compounds to be equatorially bidentate ligands to the enzyme-bound VO2+. EPR experiments with 17O-labeled P-glycolate identify the phospho group as an equatorial ligand to VO2+