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 Mn 2+ was 13.0 U/mg at 25°C. Apparent K m values at pH 7 and in the presence of 2.3 MM Mn 2+ were 0.55, 2.4 and 0.045 mM for phosphoenolpyruvate, HCO 3 and inosine 5 ′-diphosphate (IDP), respectively. Apparent K m for GDP was < 0.01 mM. ADP was not a substrate of the enzyme. Inosine 5'-triphosphate (ITP) inhibited the PEPCK activity (IC 50 = 1.7 mM), and this inhibition was not reverted by 5 mM alanine. The enzyme showed an optimum pH of 6.7 and required a divalent metal ion for activity, in the following order of effectiveness: Co 2+ > Mn 2+> Zn 2+ Mg 2+ Mg 2+ alone also activated (partially) the PEPCK. P. purpuratus adductor muscle PEPCK was inactivated by the thiol specific reagent 5,5'-dithiobis (2-nitrobenzoate) (DTNB). Titration experiments of the native enzyme suggest the presence of three thiols in the enzyme. From substrate protection experiments with IDP plus Mn 2+ and the kinetic studies of inactivation at 0°C, it is concluded that the loss of enzymatic activity with DTNB was caused by the modification of one monothiol group in the nucleotide binding region.