Alternate substrates and spectroscopic studies of P-enolpyruvate carboxykinase

P-enolpyruvate carboxykinase (PEPCK) from rat liver cytosol has been shown to catalyze the GTP-dependent phosphorylation of several α-hydroxy carboxylic acids. Glycolate, L-lactate, and L-glycerate are phosphorylated on the α-oxygen by PEPCK. The products of the reactions have been characterized by their 31P NMR spectra. Glycolate gives a higher maximal velocity than does either L-lactate or L-glycerate, and with sufficient periods of incubation GTP is quantitatively converted to GDP and the corresponding phosphorylated products in each reaction. The reactions required a divalent cation as an obligatory cofactor and the velocities of the reactions increase between pH 7 and 8. In contrast to the analogous reactions catalyzed by pyruvate kinase, PEPCK does not catalyze the phosphorylation of β-hydroxypyruvate or the bicarbonate-dependent phosphorylations of fluoride ion or hydroxylamine. Purified PEPCK normally requires μmolar concentrations of Mn(II) for full activity in the presence of mmolar Mg(II). The paramagnetic vanadyl (VO2+) ion has been shown to bind to PEPCK as a replacement for Mn(II). Saturation of the enzyme occurs near a ratio of 1.0 VO2+/enzyme catalytic site. Although VO2+ does not support catalysis, EPR experiments have shown that ternary complexes of PEPCK-VO2+ with MgGTP or the potent inhibitor oxalate are readily formed. These results show the utility of VO2+ as a paramagnetic probe in studies of the metal ion binding site of PEPCK