* To whom correspondence should be addressed.
Received April 26, 2010; Revision received May 12, 2010
The initial rates of ATP synthesis catalyzed by tightly coupled Paracoccus denitrificans plasma membrane were measured. The reaction rate was hyperbolically dependent on the substrates, ADP and inorganic phosphate (Pi). Apparent Km values for ADP and Pi were 7-11 and 60-120 µM, respectively, at saturating concentration of the second substrate (pH 8.0, saturating Mg2+). These values were dependent on coupling efficiency. The substrate binding in the ATP synthesis reaction proceeds randomly: Km value for a given substrate was independent of the concentration of the other one. A decrease of electrochemical proton gradient by the addition of malonate (when succinate served as the respiratory substrate) or by a decrease of steady-state level of NADH (when NADH served as the respiratory substrate) resulted in a proportional decrease of the maximal rates and apparent Km values for ADP and Pi (double substitution, ping-pong mechanism). The kinetic scheme for ATP synthesis was compared with that described previously for the proton-translocating ATP hydrolysis catalyzed by the same enzyme preparation (T. V. Zharova and A. D. Vinogradov (2006) Biochemistry, 45, 14552-14558).
KEY WORDS: FoF1-H+-ATP synthase, oxidative phosphorylation, bacterial plasma membrane vesicles, Paracoccus denitrificans