Creatine kinase of heart mitochondria: Changes in its kinetic properties induced by coupling to oxidative phosphorylation

A complete kinetic analysis of the forward mitochondrial creatine kinase reaction was conducted to define the mechanism for its rate enhancement when coupled to oxidative phosphorylation. Two experimental systems were employed. In the first, ATP was produced by oxidative phosphorylation. In the second, heart mitochondria were pretreated with rotenone and oligomycin, and ATP was regenerated by a phosphoenolpyruvate-pyruvate kinase system. Product inhibition studies showed that oxidative phosphorylation did not effect the binding of creatine phosphate to the enzyme. Creatine phosphate interacted competitively with both ATP and creatine, and the E · MgATP · CrP dead-end complex was not readily detected. In a similar manner, the dissociation constants for creatine were not influenced by the source of ATP: K_ib = 29 mm; K_b = 5.3 mM, and the maximum velocity of the reaction was unchanged: V₁ = 1 μmol/ min/mg. Slight differences were noted for the dissociation constant (K_ia) of MgATP from the binary enzyme complex, E · MgATP. The values were 0.75 and 0.29 mm in the absence and presence of respiration. However, a 10-fold decrease in the steady-state dissociation constant (K_a) of MgATP from the ternary complex, E · MgATP · creatine, was documented: 0.15 mm with exogenous ATP and 0.014 mm with oxidative phosphorylation. Since K_ia × K_b does not equal K_a × K_ib under respiring conditions, the enzyme appears to be altered from its normal rapid-equilibrium random binding kinetics to some other mechanism by its coupling to oxidative phosphorylation.