Paracoccus denitrificans Proton-Translocating ATPase: Kinetics of Oxidative Phosphorylation

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K. A. Kegyarikova, T. V. Zharova, and A. D. Vinogradov^*

Department of Biochemistry, Faculty of Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-1376; E-mail: adv@biochem.bio.msu.su

^* To whom correspondence should be addressed.

Received April 26, 2010; Revision received May 12, 2010

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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 (P_i). Apparent K_m values for ADP and P_i 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: K_m 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 K_m values for ADP and P_i (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).

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KEY WORDS: F_oF₁-H⁺-ATP synthase, oxidative phosphorylation, bacterial plasma membrane vesicles, Paracoccus denitrificans

DOI: 10.1134/S0006297910100081

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