The Enzymatic Carboxylation of Phosphoenolpyruvate

II. PURIFICATION AND PROPERTIES OF LIVER MITOCHONDRIAL PHOSPHOENOLPYRUVATE CARBOXYKINASE

From the ¹ From the Department of Biochemistry, New York University School of Medicine, New York, New York 10016

Phosphoenolpyruvate carboxykinase has been isolated from pig liver mitochondria and purified 208-fold from the initial mitochondrial acetone powder extract in good yield (48%). The enzyme appears homogeneous in sedimentation velocity experiments and has a sedimentation coefficient (s_20,w) of 5.21 S. Its molecular weight, determined by sedimentation equilibrium, is 73,300. The ultraviolet and visible absorption spectra of the enzyme revealed no characteristic chromophores. The amino acid composition of the enzyme is reported. At 30° mitochondrial P-enolpyruvate carboxykinase catalyzes the inosine diphosphate- and Mn⁺⁺-dependent carboxylation of 664 moles of phosphoenolpyruvate per min per mole of enzyme at pH 7.0 and the ITP- and Mn⁺⁺-dependent decarboxylation of 4650 moles of oxalacetate per min per mole of enzyme at pH 7.5. The enzyme exhibits a relatively high degree of specificity for inosine and guanosine nucleotides in the carboxylation, decarboxylation, and oxalacetate-H¹⁴CO₃^- exchange reactions. IMP, GMP, and purified ITP exhibit absolutely no activity in the carboxylation reaction; IMP and ADP are completely inactive in the decarboxylation and oxalacetate-H¹⁴CO₃^- exchange reactions. While purified IDP cannot replace ITP in the oxalacetate-H¹⁴CO₃^- exchange reaction; IDP can replace ITP in the decarboxylation reaction. In contrast to ITP-supported decarboxylation, IDP-supported decarboxylation leads to pyruvate rather than P-enolpyruvate formation. The enzyme is inhibited by low concentrations (10^-6 m) of p-chloromercuribenzoate which can be reversed by high concentrations of glutathione.

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