Substrate binding affinity changes in mitochondrial energy-linked reactions.
The effects of uncouplers and valinomycin plus nigericin (in the presence of K+) were studied on the apparent Km for substrates and apparent Vmax of the following energy-linked reactions catalyzed by submitochondrial particles: oxidative phosphorylation, NTP-33Pi exchange, ATP-driven electron transfer from succinate to NAD, and respiration-driven transhydrogenation from NADH to 3-acetylpyridine adenine dinucleotide phosphate. In all cases, partially uncoupling (up to 90%) concentrations of uncouplers of valinomycin plus nigericin were found to decrease apparent Vmax and to increase apparent Km. Results plotted as ln (Vmax/Km) versus the concentration of uncouplers or ionophores showed a linear decrease of the former as a function of increasing perturbant concentration (i.e., decreasing free energy). Because Vmax/Km may be considered as a measure of the apparent first-order rate constant for enzyme-substrate interaction and reflects the affinity between enzyme and substrate to form a complex, the results are consistent with the interpretation that membrane energization leads to a change in enzyme conformation with the resultant increase in enzyme-substrate affinity and facilitation of the reaction rate under consideration. The significance of these findings with respect to the mechanism of action of the energy-transducing systems studied is discussed.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002474:Cell-Free System; D004734:Energy Metabolism / Q000187:drug effects; D007700:Kinetics; D008929:Mitochondria, Heart / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009550:Nigericin / Q000494:pharmacology; D009711:Nucleotides / Q000378:metabolism; D010085:Oxidative Phosphorylation / Q000187:drug effects; D013379:Substrate Specificity; D014475:Uncoupling Agents / Q000494:pharmacology; D014634:Valinomycin / Q000494:pharmacology
D009711:Nucleotides; D014475:Uncoupling Agents; D009243:NAD; D014634:Valinomycin; D000255:Adenosine Triphosphate; D009550:Nigericin