A novel kinetic mechanism explaining the non-hyperbolic behavior of metal activated enzymes. Case of choline kinase from rat liver.

Abstract A novel kinetic mechanism that explains the non-hyperbolic kinetics of many metal- or effector-activated enzymes is proposed as an alternative to the allosteric, hysteresis and mnemonical models. In this mechanism, the non-Michaelian behavior is generated by a reversible binding of an essential metal cation or other effector to a single site, but to at least two different enzyme forms in steady state. The model is described by a higher degree rate equation since the metal binding to more than one enzyme form generates at least two steady-state catalytic pathways of different efficiencies in addition to the recycling of the metal-enzyme species in the kinetic sequence. The proposed mechanism also explains the transition from non-hyperbolic kinetics to a Michaelian rate law, as well as the dual activation and inhibition of enzymes by the metal cation or effector, according to its concentration. This kinetic behavior is generated by the participation of the metal or effector in fast and slow competing catalytic sequences or by the competitions produced by binding as a common reactant for both the forward and reverse reactions. The model can also explain some peculiar inhibition patterns observed for some transferases. This kinetic mechanism can be tested by an experimental protocol that includes the metal cation or effector as a controlled variable reactant. The model and its complete rate equation explains the non-Michaelian behavior of choline kinase. At low ligand concentrations, an effectively ordered terreactant sequential mechanism operates (Infant. & Kinsella, 1976). The steady-state addition of choline to free enzyme is followed by the rapid-equilibrium binding of MgATp 2− and the steady-state addition of Mg 2+ last in the sequence. Initial velocity and product inhibition studies in the non-hyperbolic kinetic region, were consistent with a partially ordered release of reactants in which phosphocholine was the first product to dissociate from the central complex. The release sequence of the other two reactants was dependent on the prevailing Mg 2+ concentration. At low Mg 2+ levels, i.e. below 2·0 mM the metal cation is predominantly released after phosphocholine whereas MgADP − is the last product to dissociate under rapid-equilibrium conditions. At higher levels of the metal cation, MgADP − is predominantly released after phosphocholine leaving the Mg-enzyme complex from which Mg 2+ may dissociate. However, a substantial fraction of the Mg-enzyme form is recycled in an alternate catalytic sequence in which the rapid-equilibrium binding MgATP 2− to the Mg-enzyme complex is followed by the steady-state addition of choline. This pathway can also be initiated by the binding of Mg 2+ to free enzyme. A third and unique sequence, which operates at low Mg 2+ concentrations, includes the participation of MgADP − as an activator via a partial reversal of one of the product release sequences. In this pathway, the rapidequilibrium binding of MgADP − to free enzyme is followed by the addition of Mg 2+ to the resulting transitory complex. Subsequent dissociation of MgADP − leaves the Mg-enzyme form, which is then channeled to product formation by the consecutive additions of MgATP 2− and choline.