Allosteric Activation of Sheep Kidney Pyruvate Carboxylase by the Magnesium Ion (Mg2+) and the Magnesium Adenosine Triphosphate Ion (MgATP2-)

The role of Mg2+ in the pyruvate carboxylase reaction has been investigated with kinetic methods. The results suggest that Mg2+ has at least two roles in the reaction mechanism; (a) it forms a complex with ATP4- to form the true MgATP2- substrate, and (b) it forms a complex with the enzyme to activate the enxymic reaction. The MgATP2- complex deviates from Michaelis-Menten kinetics in such a way as to indicate a homotropic cooperative effect involving at least 2 molecules of ligand. Excess Mg2+ induced a 3-fold stimulation of enzymic activity at saturating levels of MgATP2-. Excess Mg2+ causes changes in (a) the apparent Km value; (b) the slope of the Hill plot, and (c) the apparent Vmac when MgATP2- is the variable substrate. The apparent Km value for MgATP2- is found to be 6.6 x 10-4 m in the absence of free Mg2+ and 3.2 x 10-4 m in the presence of 1 x 10-3 m MgCl2. The apparent Km value for free Mg2+ is 2.5 x 10-4 m in the presence of 5.6 x 10-4 m MgATP2-. It is concluded that Mg2+ exhibits a heterotropic cooperative effect with respect to MgATP2-. Two possible mechanisms for the binding of Mg2+ and MgATP2- are suggested. Mg2+ may bind either at a specific effector site or, alternatively, at one of the binding sites for MgATP2- which is capable of functioning as an effector site. ATP4- was found to be a partial competitive inhibitor of MgATP2-.