Kinetics of ATP hydrolysis by F1-ATPase and the effects of anion activation, removal of tightly bound nucleotides, and partial inhibition of the ATPase by covalent modification.
Eadie-Hofstee plots (v/[S] vs. v) of the kinetics of ATP hydrolysis by purified bovine heart mitochondrial F1-ATPase (MF1) over a substrate (MgATP) concentration range of 1-5000 microM were curvilinear, indicating negative cooperativity with respect to [MgATP] as originally shown by Ebel & Lardy (1975) [Ebel, R. E., & Lardy, H. A. (1975) J. Biol. Chem. 250, 191-196]. The data were computer analyzed for the best fit of the least number of straight lines, each representing a different apparent Km and Vmax. The best fits for MF1 and TF1 from the thermophilic bacterium PS3 were three lines in each case. The upper limits of the apparent Km values for MF1 were of the order of 10(-6), 10(-4), and 10(-3) M, and the corresponding apparent Vmax values (per minute per milligram of protein) were in the range of micromoles or less for the lowest Km line and decamicromoles for the other two. The results for TF1 were very similar. The presence of an activating anion (10 mM KHCO3) in the MF1 assay medium increased the overall Vmax by about 50% and eliminated the high Km but had essentially no effect on the intermediate and low Km's, indicating retention of negative cooperativity in the corresponding substrate concentration range. Kinetic data for MgITP as substrate also yielded two Km values (in the absence of KHCO3) differing by about 10(4)-fold. The relationship between [14C]dicyclohexylcarbodiimide [( 14C]-DCCD) binding to MF1 and activity inhibition was linear up to approximately 1 mol of DCCD bound/mol of MF1. At this point, the degree of inhibition was about 95%.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000838:Anions; D001639:Bicarbonates / Q000494:pharmacology; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology*; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D000838:Anions; D001639:Bicarbonates; D046911:Macromolecular Substances; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases