Steady-state kinetics of mitochondrial nicotinamide nucleotide transhydrogenase. 2. The energy-linked reaction.

Abstract

The stedy‐state kinetics of the energy‐linked nicotinamide nucleotide transhydrogenase reaction catalyzed by submitochondrial particles from beef heart were investigated. From the relationship between reaction velocities and concentrations of substrates, patterns of product inhibition, and isotope exchange data, it is concluded that the energy‐linked trans‐hydrogenase reaction involves a ternary‐complex, Theorell‐Chance mechanism, i.e a mechanism identical to that found for the nonenergy‐linked transhydrogenase reaction. Maximal velocities of the forward and reverse reactions, Michaelis constants for the four substrates, disociation constants of the binary enzyme. substrate complexes, and rate constants of the partial reactions are calculated and compared with those found for the nonenergy‐linked reaction.The effect of energization is reflected primarily in an increase in the maximal velocity of the forword reaction and a decrease and increase, respectively of the dissociation constants of the binary enzyme. substrate and enzyme. product complexes. Inhibition of the reverse reaction upon energization is shown to be competitive with respect to the substrates NAD+ and NADPH. These findings are interpreted in terms of an energy‐ and substrate‐controlled conformational change of the enzyme between an active and an inactive form. Reaction mechanisms for the transhydrogenase reaction and its relationship to the mitochondrial energy transfer system are proposed and discussed inrelation to current hypotheses.