Synthesis of Enzyme-Bound ATP by Mitochondrial Soluble F1-ATPase in the Presence of Dimethylsulfoxide1

We found that ATP is synthesized by mitochondrial soluble F 1 -ATP from medium ADP and P 1 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 trichloroacetic 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 F 1 from ADP and P 1 in the presence of 4.2 mM Mg 2+ and 35% (w/v) DMSO at pH 6.7 and 30°C after stopping the reaction with trichloroacetic acid. The following results were obtained. The rate and extent of [α- 33 P]ATP synthesis from [α 32 P]ADP and P 1 were equal to those of [γ- 32 P]ATP synthesis from ADP and 32 P 1 . The ATP synthesized was inaccessible to hexokinase, and its amount was proportional to that of F 1 . The ATP synthesis was inhibited by sodium azide, but not by 7-chloro-4-nitro-2,1,3-benzoxadiazole, or by N , N ′-dicyclohexylcarbodiimide. No nucleoside 5′-triphosphate was synthesized by F 1 when GDP, IDP, CDP, or UDP was used as a substrate. Both the dependence on ADP concentration of the amount of ATP formed in the presence of a sufficient concentration of P 1 and the dependence on P 1 concentration of the amount in the presence of a sufficient concentration of ADP were given by the following equation: where KADP = 3 μM, Kp = 0.55 mM, and [ATP formed] max =0.4–0.6 mol/mol F 1 When the reaction mixture was diluted with the buffer solution after the ATP-synthesis 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. All these findings can be explained by the following reaction scheme in which the catalytic sites in F 1 for ATP synthesis are assumed to function independently: where the brackets indicate tight binding. However, the dependence on P 1 concentration of the initial rate of ATP synthesis, vf , in the presence of a sufficient amount of ADP was given by the equation, vf = vf·max /[1+( KP ′/{P 1 ]) 1 ]. 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 F 1 during the ATP-synthesis reaction.