Role of phosphate on the ADP-induced hysteretic inhibition of mitochondrial adenosine 5'-triphosphatase. Effects of the natural protein inhibitor.
Preincubation of F1-ATPase with ADP and Mg2+ leads to ADP binding at regulatory site inducing a hysteretic inhibition of ATP hydrolysis, i.e., an inhibition that slowly develops after Mg-ATP addition (Di Pietro, A., Penin, F., Godinot, C. and Gautheron, D.C. (1980) Biochemistry 19, 5671-5678). It is shown here that inorganic phosphate (Pi) together with ADP during preincubation abolishes the time-dependence of the inhibition after the addition of the substrate Mg-ATP. This preincubation in the presence of both Pi and ADP slowly leads to a conformation of the enzyme immediately inhibited after the addition of the substrate Mg-ATP. The Pi effect is half-maximal at 35 microM and pH 6.6, whereas a limited effect is induced at pH 8.0. The preincubation of F1-ATPase with Pi and ADP must last long enough (t1/2 = 5 min). The effects can be correlated to the amount of Pi bound to the enzyme, 1 mol Pi per mol (apparent KD of 33 microM) at saturation. Pi neither modifies the ADP binding nor the final level of the concomitant inhibition. When Pi is not present in the preincubation, the final stable rate of ADP-induced hysteretic inhibition is always reached when a near-constant amount of Pi has been generated during Mg-ATP hydrolysis. Kinetic experiments indicate that preincubation with ADP and Pi decreases both Vmax and Km which would favor a conformational change of the enzyme. Taking into account the Pi effects, a more precise model of hysteretic inhibition is proposed. The natural protein inhibitor IF1 efficiently prevents the binding of Pi produced by ATP hydrolysis indicating that the hysteretic inhibition and the IF1-dependent inhibition obey different mechanisms.
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000818:Animals; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D010710:Phosphates / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000502:physiology; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D013552:Swine
D010710:Phosphates; D011480:Protease Inhibitors; D000244:Adenosine Diphosphate; D006180:Proton-Translocating ATPases