Phosphoenolpyruvate carboxykinase from ribbed mussel gill tissue: Reactivity with metal ions, kinetics, and action of 3‐mercaptopicolinic acid

Abstract

AbstractPhosphoenolpyruvate carboxykinase (E.C. 4.1.32) from ribbed mussel gill cytosol exists as a monomer with Mr 70,000. Low levels of Mn2+ or Zn2+ were required for activity when assayed at high physiological Mg2+ levels. Mg2+ alone would not activate the enzyme. Optimal activity was observed at pH 6.6. The apparent Kms for phosphoenolpyruvate (PEP) and NaHCO3 were 396 μM and 13 mM, respectively, when assayed at pH 6.8 in the presence of 1 mM Mn2+, 1 mM Mg2+, and 1.5 mM IDP. GDP (Kmapp 24 μM) and IDP (Kmapp 46 μM) were both reactive; other nucleoside diphosphates were not. Under standard assay conditions at pH 6.8, the reaction was freely reversible with the rate of PEP carboxylation two to three times the rate of OAA decarboxylation. When assayed in the PEP synthesizing direction at pH 6.8 with 2 mM Mn2+ and Mg2+, the apparent Kms for GTP and OAA were 55 μM and 24 μM, respectively. Chloride ion was inhibitory (I50 120 mM); 3‐mercaptopicolinic acid (MPA) and 3‐aminopicolinic acid (APA) were competitive inhibitors with respect to PEP and noncompetitive with respect to IDP. MPA was a more effective inhibitor (Kiapp 8–34 μM) than APA (Kiapp 250–500 μM). Aminooxyacetate and N‐butylmalonate (BMA) were not inhibitory and hydroxymalonate was slightly inhibitory (Kiapp 15 mM). ATP showed mixed competitive inhibition with respect to PEP (Kiapp 0.54 mM); inhibition by ATP was not reversed by L‐alanine. Experiments employing BMA and MPA with gill tissue pieces subjected to hyperosmotic stress indicated that the PEPCK reaction in gill tissue was not significantly involved in the regulation of carbon flow for the synthesis of L‐alanine as an intracellular osmotic buffer.