Activation/Inactivation Equilibrium of CF₁-ATPase

O. I. Vitseva¹ and A. N. Malyan^1,2

¹Institute of Soil Science and Photosynthesis, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (7-0967) 79-0532; E-mail: Malyan@issp.serpukhov.su

²To whom correspondence should be addressed.

Submitted February 10, 1997; revision submitted March 12, 1997.

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Chloroplast coupling factor (CF₁-ATPase) is reversibly inactivated on incubation with magnesium ions. The inactivation has two stages: the fast stage (k = 6 min^-1) occurs at high pH, while the slow stage (k = 0.1-0.2 min^-1) depends on pH only slightly over the entire studied range (5.5-9.0). The varying inactivation effect of Mg²⁺ suggests its affinity for the enzyme decreases dramatically with decreasing pH of the medium. The pH dependence of the apparent dissociation constant of Mg²⁺ suggests that the equilibrium between Mg²⁺ binding and dissociation is determined by protonation/deprotonation of specific acid-base groups of the enzyme. Analysis of the dependence plots yielded an equilibrium constant of magnesium dissociation (3-9 µM) and the dissociation constant of protonated groups of the enzyme (pK = 5.8-6.7). Addition of MgADP--CF₁ complex-stabilizing sodium azide to the incubation medium lowered the dissociation constant of Mg²⁺ and produced no effect on the dissociation constant of acid-base groups. Reactivation of Mg²⁺-inactivated CF₁-ATPase occurs on decreasing the pH of the medium. The reactivation rate increases in the presence of micromolar concentrations of ATP or octyl glucoside and changes only slightly with ADP or Triton X-100. These results suggest that reactivation of CF₁-ATPase is determined by protonation of a limited number of acid-base groups submerged in the enzyme molecule.

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KEY WORDS: chloroplast coupling factor, H⁺-ATPase, activation/inactivation, pH dependence, Mg²⁺ binding, acid-base groups, ATP hydrolysis.

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