Synthesis of Enzyme-Bound ATP by Mitochondrial Soluble F₁-ATPase in the Presence of Dimethylsulfoxide

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We found that ATP is synthesized by mitochondria) soluble F₁-ATPase from medium ADP and Pi in the presence of dimethylsulfoxide (DMSO), with the amount synthesized increasing with the DMSO concentration to a maximum at 30% (w/v) DMSO. In the presence of 35% (w/v) DMSO, ADP was scarcely converted into AMP and much more ATP was formed than AMP. The pH dependence curve of ATP synthesis was bell-shaped with the optimum at 6.7. The amount of synthesized ATP measured after stopping the reaction with triehloroacetic acid was almost equal to that measured after stopping the reaction with sodium dodecyl sulfate or with ethanol. Therefore, we measured the amount of ATP synthesized by F₁ from ADP and P₁ in the presence of 4.2mM Mg²⁺ and 35% (w/v) DMSO at pH 6.7 and 30°C after stopping the reaction with triehloroacetic acid. The following results were obtained.
1. The rate and extent of [α-³²P] ATP synthesis from [α-³²P] ADP and P, were equal to those of [γ-³²P]ATP synthesis from ADP and ³²P₁.
2. The ATP synthesized was inaccessible to hexokinase, and its amount was proportional to that of F, The ATP synthesis was inhibited by sodium azide, but not by 7-chloro-4-nitro-2, 1, 3-benzoxadiazole, or by N, N'-dicyclohexylcarbodiimide.
3. No nucleoside 5'-triphosphate was synthesized by Fl, when GDP, IDP, CDP, or UDP was used as a substrate.
4. Both the dependence on ADP concentration of the amount of ATP formed in the presence of a sufficient concentration of Pi and the dependence on P₁ concentration of the amount in the presence of a sufficient concentration of ADP were given by the following equation:
[ATP formed]=[ATP formed]_max/(1+K_ADP/[ADP] or Kp/[P₁])
where K_ADP=3 μM, K_p=0.55mM, and [ATP formed]_max=0.4-0.6 mol/mol F₁.
5. When the reaction mixture was diluted with the buffer solution after the ATPsynthesis reaction had reached equilibrium, the amount of synthesized ATP decreased monophasically at a higher rate than that of ATP formation. When the pH of the reaction mixture was rapidly increased from 6.9 to 8.0, about half of the synthesized ATP disappeared very rapidly, while the remainder decreased rather slowly.
6. All these findings can be explained by the following reaction scheme in which the catalytic sites in F₁ for ATP synthesis are assumed to function independently:
_??_
where the brackets indicate tight binding.
7. However, the dependence on P₁ concentration of the initial rate of ATP synthesis, v_f, in the presence of a sufficient amount of ADP was given by the equation, v_f=V_f•max/[1+(Kp'/[P₁])²]. Furthermore, when AMPPNP was added to the reaction mixture, 60-70% of the formed ATP disappeared very rapidly and the remainder decreased very slowly. These two findings suggest cooperativity between catalytic or nucleotide-binding sites of F₁ during the ATP-synthesis reaction.