Implications of the Existence of Two States of Beef Liver Mitochondrial Adenosine Triphosphatase as Revealed by Kinetic Studies

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Abstract
The results of studies on the initial velocity in hydrolysis reactions of ATP and other nuclcoside triphosphates with beef liver mitochondrial ATPase can be summarized as follows. 1. Double reciprocal plots of substrate concentration vs. initial velocity were linear for all the nucleoside triphosphates tested except for ATP, which showed a negative cooperativity. 2. Bicarbonate ion increased the rate of ATP hydrolysis and diminished its negative cooperativity, whereas hydrolysis of other nucleoside triphosphates was only slightly affected by the anion. 3. An excess of nucleoside triphosphate apparently inhibited its own hydrolysis for all kinds of nucleoside triphosphates tested, whereas an excess of magnesium ion apparently inhibited only ATP hydrolysis. 4. Inhibition of ATPase activity with an excess of magnesium ion was no longer observed when the reaction was carried out at low temperature (10°C) or in the presence of sulfate. Under these conditions, the kinetics of ATP hydrolysis were apparently of simple Michaelis-Menten type. These observations suggest the existence of two states (“A” and “N”) of beef liver mitochondrial ATPase. The state “A” is characterized by phenomena specifically affecting ATP hydrolysis, such as the inhibition by excess magnesium ion, and the negatively cooperative profile in the dose-response curve of ATP. In the state “N” proceed the hydrolysis reactions of other nucleoside triphosphates and of ATP under limited conditions. The two states (“A” and “N”) can be related to an enzyme model with a catalytic site and a regulatory site. Computor simulation revealed that such a model could account well for the experimental data.

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