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

Abstract

1. 1. Dinitrophenol and maleate anions increase V ATP 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. 2. Preincubation with ATP, iso-GTP or 2′-deoxy ATP results in a metastable enzyme form with a raised V and a reduced K m. Dinitrophenol stimulates both ATP and ITP hydrolyses by this form. 3. 3. The Arrhenius plot of ATP (but not ITP) hydrolysis by the isolated ATPase shows a break at about 18°C, apparently because the rate limiting step of hydrolysis changes as the temperature rises. 4. 4. Adenylyl β,γ-imidodiphosphate (Ado PP[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 Ado PP[NH] P binding increases. It is concluded that Ado PP[NH] P inhibits by binding to the hydrolytic site of the enzyme. 5. 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.