Kinetic mechanism of phosphoenolpyruvate carboxykinase (GTP) from rat liver cytosol. Product inhibition, isotope exchange at equilibrium, and partial reactions.
Initial velocity studies of rat liver cytosolic P-enolpyruvate carboxykinase in the direction of P-enolpyruvate formation gave intersecting double reciprocal plots indicating that the reaction conforms to a sequential reaction pathway. A complete product inhibition study with MnGDP-, P-enolpyruvate, and HCO3- as product inhibitors indicated that all patterns were noncompetitive. Isotope exchange at equilibrium with exchange between the substrate/product pairs GTP/GDP oxalacetate/HCO3-, and oxalacetate/P-enolpyruvate while varying the concentration of substrate/product pairs in fixed constant ratio gave no complete inhibitory patterns as the concentration of the constant ratio pairs approached saturation. The exchange rates between the substrate/product pairs differed by a factor of 40 when compared under the same assay conditions. These results were interpreted in terms of a random  reaction mechanism in which true dead-end complexes do not form and in which the rate-limiting step is not the interconversion of the ternary quarternary central complexes. In addition to the formation of P-enolpyruvate from oxalacetate and MnGTP2-, the enzyme catalyzes the decarboxylation of oxalacetate to pyruvate in the absence of MnGTP2-. This reaction occurs only slowly in the absence of GDP and most rapidly in the presence of MnGDP-. When only MnGTP2- and oxalacetate are present, no pyruvate is formed, and oxalacetate is converted stoichiometrically to P-enolpyruvate. The enzyme also catalyzes the exchange of [14C]GDP into GTP in the absence of P-enolpyruvate. This exchange is stimulated by the presence of HCO3-. When enzyme is incubated with MnGTP2- in the presence or absence of HCO3-, there is no hydrolysis to form GDP and P1. The two partial reactions, namely the exchange of [14C]GDP with the E.HCO3.MnGTP or E.MnGTP complex and the formation of pyruvate from the E.oxalacetate.MnGDP complex provide pathways by which the expected dead-end complexes can be converted to enzyme forms which can return to the catalytic or exchange sequence.
D000818:Animals; D002250:Carbon Radioisotopes; D003600:Cytosol / Q000201:enzymology; D006160:Guanosine Triphosphate; D007553:Isotope Labeling; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism*; D051381:Rats
D002250:Carbon Radioisotopes; D006160:Guanosine Triphosphate; D010729:Phosphoenolpyruvate Carboxykinase (GTP)