Pedersen P L
J Supramol Struct. 1975;3(3):222-30. doi: 10.1002/jss.400030304.
Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, KD = 1-2 muM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (KI = 240-300 muM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This "tightly bound" ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetics studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, Pi can maintain the active form of the enzyme.
大鼠肝脏线粒体的线粒体ATP酶含有多个核苷酸结合位点。在低浓度下，ADP以高亲和力结合（1摩尔/摩尔ATP酶，KD = 1 - 2μM）。在高浓度下，ADP可能通过与ATP竞争活性位点来抑制ATP水解（KI = 240 - 300μM）。刚分离出来时，线粒体ATP酶每摩尔ATP酶含有0.6至2.5摩尔ATP。这种“紧密结合”的ATP可以通过用硫酸铵反复沉淀去除，而不会改变酶的水解活性。然而，ATP耗尽的酶必须重新溶解在高浓度的磷酸盐中以保持活性。AMP - PNP（腺苷酰亚胺二磷酸）取代从酶中去除的紧密结合的ATP并抑制ATP水解。AMP - PNP对ADP的高亲和力结合几乎没有影响。ATP水解的动力学研究表明，只要在碳酸氢盐缓冲液或含有高浓度磷酸盐的缓冲液中进行测定，速度与ATP的关系曲线呈双曲线。综上所述，这些研究表明，酶上不直接与ATP水解相关的位点结合ATP或ADP，并且在没有结合核苷酸的情况下，Pi可以维持酶的活性形式。
