Studies of the kinetics of the isolated mitochondrial ATPase using dinitrophenol as a probe

1. Dinitrophenol and maleate anions increase VATP on the 'washed', isolated, mitochondrial ATPase. Hydrolyses of iso-GTP and 2'-deoxy ATP are also stimulated, while hydrolyses of other nucleoside triphosphates (ITP, GTP etc.) are not. 2. Preincubation with ATP, iso-GTP or 2'-deoxy ATP results in a metastable enzyme form with a raised V and a reduced Km. Dinitrophenol stimulates both ATP and ITP hydrolyses by this form. 3. The Arrhenius plot of ATP (but not ITP) hydrolysis by the isolated ATPase shows a break at about 18 degrees C, apparently because the rate limiting step of hydrolysis changes as the temperature rises. 4. Adenylyl beta, gamma-imidodiphosphate (AdoPP[NH]P) inhibits ITP hydrolysis in a pseudofirst order reaction. Its binding is competitive with ITP. If the enzyme is preincubated with ATP, the rate of AdoPP[NH]P binding increases. It is concluded that AdoPP[NH]P inhibits by binding to the hydrolytic site of the enzyme. 5. We conclude that ATP hydrolysis is limited by diphosphate release and ITP hydrolysis by bond splitting. Energy release during ATP hydrolysis is maximal at the ATP binding step, and during ITP hydrolysis at bond splitting.