Synthesis of Medium Pyrophosphate by Soluble Mitochondrial F1 through Dimethyl Sulfoxide-Water Transitions

Abstract

Soluble F1 from heart mitochondria incubated in mixtures that have Mg2+, inorganic phosphate, and dimethyl sulfoxide (40% (v/v)) catalyzes the spontaneous synthesis of ATP and pyrophosphate (Tuena de Gómez-Puyou, M., García, J. J., and Gómez-Puyou, A. (1993) Biochemistry 32, 2213-2218). By filtration techniques, it was determined that synthesized ATP and pyrophosphate are enzyme bound, albeit the affinity for pyrophosphate was lower than that of ATP. After ATP and pyrophosphate were formed in dimethyl sulfoxide mixtures, dilution with aqueous buffer to a dimethyl sulfoxide concentration of 6.0% brought about the partition of pyrophosphate into the media. This was evidenced by filtration experiments as well as by the accessibility of synthesized pyrophosphate to soluble inorganic pyrophosphatase. Release of pyrophosphate induced by dilution occurred in less than 15 s. Under conditions that produce release of pyrophosphate, no release of ATP was observed; instead, ATP underwent hydrolysis. Studies on the effect of arsenate on the synthesis and hydrolysis of ATP and PPi in F1 showed that hydrolysis of synthesized PPi at its site of synthesis was slower than that of ATP. Thus, the question of whether differences in the rates of hydrolysis accounted for the dilution-induced release of PPi but not of ATP was addressed. Synthesis and hydrolysis of ATP and pyrophosphate were examined in preparations of soluble F1 in complex with its inhibitor protein; the complex had an ATPase activity about 100 times lower than that of free F1. In mixtures that contained dimethyl sulfoxide, the complex synthesized ATP and pyrophosphate at nearly the same rates; upon dilution, hydrolysis of both compounds occurred also at similar rates, yet only pyrophosphate was released. The same phenomenon was observed in F1 that had been depleted of adenine nucleotides. Hence, dilution-induced release of PPi was independent of the overall catalytic properties of the enzyme or its content of adenine nucleotides. Since synthesis of ATP occurs at the expense of the ADP that remains after depletion of adenine nucleotides, it is likely that the failure of ATP to be released is due to the high affinity that F1 exhibits for the synthesized ATP. Nevertheless, the results illustrate that a complete catalytic cycle that starts with medium Pi and ends with medium pyrophosphate may be reproduced in soluble mitochondrial F1.

Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase

“…ATP was not formed when the enzyme was in 30 % (v\v) DMSO. This contrasts with the published behaviour of the F " -ATPase [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]31]. The exchange of exogenous phosphate into bound phosphate, the phosphate-ATP exchange reaction and the formation of ATP in a DMSO-free buffer were insensitive to azide.…”

“…The remaining four molecules of phosphate could be bound at the two unoccupied nucleotide-binding sites. That two molecules of phosphate can occupy a single nucleotide-binding site is suggested by the work of Tuena de Gomez-Puyou et al [17,18], who found that pyrophosphate was made by F " in the presence of DMSO. However, it is also possible that some phosphate is bound at other, non-nucleotide-binding, sites.…”

“…In contrast to F " [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23], the F " F o -ATP synthase did not form ATP from ADP and phosphate in the presence of DMSO. However, pretreatment with DMSO, followed by removal of this compound on centrifuged columns of Sephadex G-50 was necessary to induce net formation of ATP.…”

“…Both methods were equally suitable for the assay of F " F o -ATP synthase protein concentrations in DMSOfree samples. Pyrophosphate was assayed by the extraction method of Tuena de Gomez-Puyou et al [17,18]. The relative mass of the F " F o -ATP synthase used in our calculations was 633 400 Da [10].…”

“…Since the F " -ATPase in 30 % (v\v) DMSO-containing buffers was able to synthesize ATP from ADP and P i [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27], the effect of adding ADP to [$#P]P i -loaded F " F o -ATP synthase in 30 % (v\v) DMSO was examined. As shown in Figure 3, ADP caused Effect of DMSO on ATP synthase the release of 1 mol of P i \mol of F " F o -ATP synthase.…”