Purification and characterization of pyruvate carboxylase from Arthrobacter globiformis

Abstract

Pyruvate carboxylase from Arthrobacter globiformis has been purified approximately 300-fold. The highly purified enzyme, with a specific activity of 27.0 units/mg of protein, gives two biotin-containing protein bands on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, which constitute about 80% of the total protein. The enzyme requires pyruvate, MgATP, and HCO 3 − for activity, is activated by univalent and divalent cations, and is inhibited by avidin. The enzyme has about 4% of its maximum activity in the absence of acetyl-CoA, which activates the enzyme with an A 0.5 value of 25 μ m. Activation is sigmoidal, with a Hill coefficient of above 3.0, and is antagonized by l-aspartate, which has a Hill coefficient of between 2.0 and 4.0 depending on the acetyl-CoA concentration. The enzyme is inactivated at 0 °C or at pH 8.0 (15 °C), at low salt concentrations with a concomitant change in sedimentation velocity, indicating a halving of molecular weight. The molecular weight of the inactive form of the enzyme formed at pH 8.0 is 290,000, implying a value of about 580,000 for the molecular weight of the native enzyme. The subunit molecular weight of the enzyme was shown by polyacrylamide gel electrophoresis in sodium dodecyl sulfate to be 130,000, suggesting that the native enzyme is tetrameric. This suggestion is supported by the fact that the sedimentation coefficient of the native enzyme ( s 20,w = 15.0 S), is similar to that of several tetrameric pyruvate carboxylases from other sources. The results show that the A. globiformis pyruvate carboxylase has regulatory properties in common with the enzyme from several other sources but has several unique structural characteristics.