PURIFICATION, PARTIAL KINETIC CHARACTERIZATION AND REACTIVE SULFHYDRYL-GROUPS OF THE PHOSPHOENOLPYRUVATE CARBOXYKINASE FROM PERUMYTILUS-PURPURATUS ADDUCTOR MUSCLE

Phosphoenolpyruvate carboxykinase (PEPCK) from the adductor muscle of Perumytilus purpuratus was purified to homogeneity, as determined by S DS-polyacrylamide gel electrophoresis (PAGE), The purification consist ed of a three-step procedure: ammonium sulphate precipitation, ion exc hange chromatography on phosphocellulose and affinity chromatography o n 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 degrees C, Apparent K-m values at pH 7 and in the presence of 2.3 mM Mn2+ were 0.55, 2.4 and 0.045 mM for phosphoenolpyruvate, H CO3- 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'-t riphosphate (ITP) inhibited the PEPCK activity (IC50 = 1.7 mM), and th is inhibition was not reverted by 5 mM alanine. The enzyme showed an o ptimum pH of 6.7 and required a divalent metal ion for activity, in th e following order of effectiveness: Co2+ > Mn2+ > Zn2+ > Mg2+. Mg2+ al one also activated (partially) the PEPCK, P. purpuratus adductor muscl e PEPCK was inactivated by the thiol specific reagent 5,5'-dithiobis ( 2-nitrobenzoate) (DTNB). Titration experiments of the native enzyme su ggest the presence of three thiols in the enzyme, From substrate prote ction experiments with IDP plus Mn2+ and the kinetic studies of inacti vation at 0 degrees C, it is concluded that the loss of enzymatic acti vity with DTNB was caused by the modification of one monothiol group i n the nucleotide binding region.