Relationships of inosine triphosphate and bicarbonate effects on F₁ ATPase to the binding change mechanism

Abstract

Two interesting previously reported properties of mitochondrial F₁ ATPase have been confirmed and have been examined by¹⁸O exchange measurements to assess if they are consistent with sequential participation of catalytic sites during ATP hydrolysis. These are the ability of HCO ⁻₃ to increase reaction rate with apparent loss of cooperative interaction between subunits and the ability of ITP to accelerate the hydrolysis of a low concentration of ATP. The effect of HCO ⁻₃ was tested at concentrations of ATP lower than previous measurements. The activation disappeared when ATP was reduced to 0.1 µM. The HCO ⁻₃ activation at higher ATP concentrations did not change the extent of reversal of the cleavage of tightly bound ATP at the catalytic site, as measured by the average number of water oxygens incorporated with each P_i formed when 5 or 10 µM ATP is hydrolyzed. The data are consistent with sequential site participation with HCO ⁻₃ acceleration of ADP departure after a binding change that stops¹⁸O exchange and loosens ADP binding.

When ITP concentration was lowered during net ITP hydrolysis by F₁ ATPase an increase in water oxygen incorporation into P_i formed is observed, as noted previously for ATP hydrolysis. The acceleration of the cleavage of a constant low concentration of [γ-¹⁸O]ATP by concomitant hydrolysis of increasing concentrations of ITP was accompanied by a decrease in water oxygen incorporation with each P_i formed from the ATP. These results add to evidence for the binding change mechanism for F₁ ATPase with sequential participation of catalytic sites.