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The purification, characterization, and activation of phosphoenolpyruvate carboxykinase from chicken liver mitochondria.

C. Hebda,T. Nowak

Published 1982 in Journal of Biological Chemistry

ABSTRACT

The enzyme phosphoenolpyruvate carboxykinase has been purified from chicken liver mitochondria. This purification includes a pseudo-affinity column step utilizing Sepharose 4B-blue dextran which binds the enzyme. The enzyme elutes with ITP to yield protein which is greater than 98% pure. The enzyme has Mr = 75,400 +/- 200 estimated by high speed sedimentation equilibrium and 70,500 +/- 500 estimated by reduced sodium dodecyl sulfate-polyacrylamide gels. The enzyme is abnormally retarded on molecular exclusion resins yielding low apparent molecular weight values. The amino acid analysis indicates that the enzyme has a high proline content and a high tryptophan content and contains 9 mol of cysteine/mol of enzyme. No disulfide bonds were detected. The extinction coefficient (epsilon 1% 280 = 16.5 +/- 0.1) reflects the high tryptophan content. The Svedberg coefficient (s20,w = 4.63 +/- 0.03 S) is consistent with a globular protein of Mr = 70,500-75,400. The activation of the enzyme was investigated by steady state kinetics. The carboxylation reaction has an activation energy of 17.6 kcal/mol. There is no requirement of a monovalent cation for activity. A thiol is necessary for maximal activity, although apparently not to reduce disulfide bonds within the enzyme. Incubation with dithiothreitol stabilizes enzymatic activity but beta-mercaptoethanol facilitates loss of activity. The kinetics of activation by Mn2+ was performed. The Ks value for phosphoenolpyruvate (300 microM) decreases to an apparent Km of 67 microM with increasing concentrations of Mn2+. The concentration of Mn2+ does not affect the interaction of HCO-3 with the enzyme, however. Analysis of data in terms of free IDP indicates that increasing Mn2+ decreases the Km of IDP but analysis as MnIDP indicates the Km,app of MnIDP is independent of the Mn2+ concentration. The enzyme interacts with Mn2+ with a KA = 67 microM and the Km,app decreases to a value of 8 microM with saturating substrates. The substrate analogue (Z)-3-fluorophosphoenolpyruvate is a good substrate for the reaction (Km = 30 microM) with 27% Vmax compared to P-enolpyruvate (Km = 180 microM). Except for 3-bromophosphoenolpyruvate, other analogues have shown weak competitive or noncompetitive inhibition. Potential analogues of oxalacetate (succinate, citrate, isocitrate, malate, and alpha-ketoglutarate) all elicit weak (greater than 15 mM) inhibition.

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