Acceleration of Unisite Catalysis of Mitochondrial F₁‐Adenosinetriphosphatase by ATP, ADP and Pyrophosphate

Abstract

The effect of ATP, ADP and pyrophosphate (PP,) on hydrolysis and release of [y-'*P]ATP bound to the high-affinity catalytic site of soluble F, from bovine heart mitochondria under unisite conditions [Grubmeyer, C., Cross, R. L. & Penefsky, H. S. (1982) J. B i d . Chem. 257, 12092-121001 was studied. In accord with the previous data, it was observed that millimolar concentrations of ATP or ADP added to F, undergoing unisite hydrolysis of [y-"PIATP accelerated its hydrolysis. PP, also produced a hydrolytic burst of a fraction of the previously bound [y-izP]ATP; kinetic data suggested that for production of optimal hydrolysis by PP, of the bound [y-'*P]ATP, two binding sites with apparent Kd of 27 pM and 240 pM must be filled. The extent of the hydrolytic burst induced by MgPP, was lower than that induced by ADP and ATP. In F, in which PP, had produced a hydrolytic burst of the bound [y-3zP]ATP, the addition of ATP induced a second burst of hydrolysis. By filtration experiments and enzyme trapping, it was also studied whether ATP, ADP and PP, produce release of the tightly bound [y-'*P]ATP. At millimolar concentrations, ATP and ADP brought about release of about 25 % of the previously bound [y-"PIATP. At micromolar concentrations, ADP accelerated the hydrolysis of the previously bound [Y-~'P]ATP but not its release. Hence, the hydrolytic and release reactions could be separated, indicating that the two reactions require the occupancy of different sites in F,. With PP,, no release of the tightly bound [y-?'P]ATP was observed. The ADP induced hydrolysis and release of the F,-bound [y-'*P]ATP were inhibited by sodium azide to the same extent (60%). Since release of ATP from a high-affinity catalytic site of F, represents the terminal step of oxidative phosphorylation, the data illustrate that the binding energy of substrates to F, is critical to the ejection of ATP into the media. The failure of PP, to induce release of [y-"P]ATP bound to F, under unisite conditions is probably due to its lower binding energy.

Synthesis and hydrolysis of ATP and the phosphate–ATP exchange reaction in soluble mitochondrial F1 in the presence of dimethylsulfoxide

“…5A). In water, confirming previously published data [31], the same results were obtained (data not shown).…”

Regulation of the F1F0-ATP Synthase Rotary Nanomotor in its Monomeric-Bacterial and Dimeric-Mitochondrial Forms

“…ATP synthesis occurs in the whole F 1 F 0 when the energy derived from proton conduction through the F 0 membrane channel is combined with the nucleotide (Mg-ADP) and Pi binding energies [2][3][4][5] to drive the release of newly synthesized ATP from each of the three alternating catalytic sites of F 1 . The coupling between F 1 and F 0 is critical for efficient ATP synthesis to occur and major progress in the understanding of this coupling mechanism has been achieved.…”

Inorganic pyrophosphate generation and disposition in pathophysiology

“…The ability of PP i , like other phosphate esters, to serve as an intracellular biochemical intermediate may be underestimated in higher organisms (17). For example, at physiological concentrations, PP i can interact with and potentially modify the activity of certain ATPases, e.g., myosin magnesium ATPase and F 1 -ATPase (10,33,68). PP i also facilitates iron transfer between transferrin and ferritin and iron delivery to mitochondria from transferrin (20).…”

“…PP i also facilitates iron transfer between transferrin and ferritin and iron delivery to mitochondria from transferrin (20). Certain previous overviews of PP i in mitochondrial metabolism merit attention (33,68) for discussions of the ability of PP i to function in energy transfer reactions in yeast, where PP i , in combination with inorganic pyrophosphatase, generates a mitochondrial membrane potential (88). ATP-derived PP i also can function as a phosphate donor in protein phosphorylation in not only yeast mitochondria but also in mammalian cells (21).…”