The pre-steady state and steady-state kinetics of the ATPase activity of mitochondrial F1.
1. The lag time before maximum velocity of ATP hydrolysis is reached upon mixing ATP with F1 is much greater than can be explained by a simple Michaelis-Menten mechanism, and must be due to an activation reaction. The lag time is dependent on the concentration of MgATP (half-maximal at 30 microM) and is equal to 30 ms at infinite MgATP concentration. The initial rate of hydrolysis by nucleotide-depleted F1 is much greater than with normal F1. It is tentatively suggested that the activation reaction with normal preparations is due to replacement of firmly bound ADP by MaATP. 2. After the initial time lag, the reaction follows very closely first-order kinetics provided that the concentration of MgATP is much less than the Km and the reaction is completed within 2 s. This is not expected if the dissociation constant of the enzyme-MgADP complex, an intermediate in the enzymic reaction, is much lower than the Km as has been reported in the literature. The value of V/Km, calculated from the exponential decay, is very close to that calculated from independent measurements of V and Km. 3. The low values for Ki(ADP) reported in the literature were found to be due to a slow (in the order of seconds) formation of an inhibited MgADP-enzyme complex. Dissipation of this inhibited complex by ATP requires seconds. The dissociation constant of the MgADP-enzyme complex that is an intermediate in the enzyme reaction was found to be 150 microM. 4. ADP but not ATP becomes firmly bound to nucleotide-depleted F1 in the absence of Mg2+.
D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010086:Oxidative Phosphorylation Coupling Factors / Q000378:metabolism*; D006180:Proton-Translocating ATPases
D010086:Oxidative Phosphorylation Coupling Factors; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; D008274:Magnesium