Requirement of medium ADP for the steady-state hydrolysis of ATP by the proton-translocating Paracoccus denitrificans F o·F 1-ATP synthase

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F o·F 1-ATP synthase in inside-out coupled vesicles derived from Paracoccus denitrificans catalyzes P i-dependent proton-translocating ATPase reaction if exposed to prior energization that relieves ADP·Mg 2+-induced inhibition (Zharova, T.V. and Vinogradov, A.D. (2004) J. Biol. Chem., 279, 12319–12324). Here we present evidence that the presence of medium ADP is required for the steady-state energetically self-sustained coupled ATP hydrolysis. The initial rapid ATPase activity is declined to a certain level if the reaction proceeds in the presence of the ADP-consuming, ATP-regenerating system (pyruvate kinase/phospho enol pyruvate). The rate and extent of the enzyme de-activation are inversely proportional to the steady-state ADP concentration, which is altered by various amounts of pyruvate kinase at constant ATPase level. The half-maximal rate of stationary ATP hydrolysis is reached at an ADP concentration of 8 × 10 −6 M. The kinetic scheme is proposed explaining the requirement of the reaction products (ADP and P i), the substrates of ATP synthesis, in the medium for proton-translocating ATP hydrolysis by P. denitrificans F o·F 1-ATP synthase.