Purification, partial kinetic characterization and reactive sulfhydryl groups of the phosphoenolpyruvate carboxykinase from Perumytilus purpuratus adductor muscle

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) from the adductor muscle of Perumytilus purpuratus was purified to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis (PAGE). The purification consisted of a three-step procedure: ammonium sulphate precipitation, ion exchange chromatography on phosphocellulose and affinity chromatography on GTP-agarose. The enzyme presented a native molecular mass of 85 kDa, appearing as an active monomer. Under denaturing conditions (SDS-PAGE), the enzyme showed a relative molecular mass of 74 kDa. The specific activity of homogeneous PEPCK in the presence of 2.3 mM Mn2+ was 13.0 U/mg at 25°C. Apparent Km values at pH 7 and in the presence of 2.3 MM Mn2+ were 0.55, 2.4 and 0.045 mM for phosphoenolpyruvate, HCO3 and inosine 5′-diphosphate (IDP), respectively. Apparent Km for GDP was < 0.01 mM. ADP was not a substrate of the enzyme. Inosine 5'-triphosphate (ITP) inhibited the PEPCK activity (IC50 = 1.7 mM), and this inhibition was not reverted