Structural rearrangements in soluble mitochondrial ATPase.

Treatment of isolated factor F1 by 1% dimethylsuberimidate in the presence of 50 mM (NH4)2SO4 leads to the formation of four different types of cross-linked dimers of the subunits, on average one dimer per molecule of the enzyme. This treatment results in 60-70% inactivation of factor F1. Factor F1 treated with dimethylsuberimidate does not show a change in the sedimentation coefficient and is not inactivated in the cold; it is not inactivated in the presence of Mg2+ either, nor is it activated by anions. Incubation of the cross-linked factor F1 with ADP does not lead to inactivation, although the ability to tightly bind ADP is retained. The total quantity of tightly bound ADP reaches 5 mol per mol of the cross-linked factor F1. Cross-linking of factor F1 also prevents the slow inactivation of the enzyme coupled with the hydrolysis of Mg-ATP and Mg-GTP. The dependence of the inactivation rate constant on the concentration of Mg-ATP and Mg-GTP at substrate concentrations of 0.05-2 mM is characterized by the same values of Km,app as those of the ATPase and GTPase activities of factor F1. The probability of the inactivation of factor F1 per turnover remains constant for all the concentrations of the substrates studied and is 2 . 10(-6) per turnover for the ATPase reaction and 2 . 10(-5) per turnover for the GTPase reaction. Moderate hydrostatic pressure (up to 150 atmospheres) greatly accelerates ATP-induced inactivation of factor F1. The activation volume (delta V*) of the inactivation process is equal to 5.1 . 10(-4) cm3/g, which is evidence of considerable changes in the extent of protein hydration during inactivation. Inactivation of the enzyme under pressure is accompanied by dissociation into subunits. Dimethyladipimidate, which does not cause intersubunit cross-linking in the molecule of factor F1, does not alter the properties of the native enzyme. It is suggested that the formation of one intersubunit cross-link in the molecule of factor F1 by dimethylsuberimidate affects the ability of the enzyme to undergo co-operative rearrangements of the quaternary structure under the influence of Mg2+, ADP, ATP, anions, and low temperature. The rate constants of ATP binding to the active site of factor F2 (k+1) = 2 . 10(8) M-1 . min-1), of ATP release from the active site (k-1 = 2 . 10(-2) min-1), and of ADP and Pi release from the active site (k2 = 5 . 10(3) min-1) have been determined. The results obtained confirm the correctness of Boyer's idea, according to which ATP is formed in the active site of mitochondrial ATPase without any external source of energy. Energy is used at the stage of the release of synthesized ATP from the active site of ATPase in the solution.

Investigation of soluble mitochondrial ATPase by the reacting enzyme sedimentation method.

Soluble mitochondrial ATPase from bovine heart (factor F1) loses its activity during ATP hydrolyses. The inactivation is accelerated by moderate pressure, which is generated in an ultracentrifuge cell. The rate of inactivation slows down if the concentration of the substrate (MgATP) is diminished. ATP hydrolysis proceeds at an almost constant rate if the substrate concentration is as low as 0.05 mM. One intersubunit cross-link formed by dimethylsuberimidate per molecule of factor F1, prevents its inactivation during the ATPase reaction both without pressure and in an ultracentrifuge. Sedimentation coefficients measured by the reacting enzyme centrifugation method of both unmodified factor F1 at a low (about 0.05 mM MgATP) substrate concentration and of its dimethylsuberimidate cross-linked form in the presence of 10 mM MgATP, were determined to be s20, w = 12.4 +/- 0.4 S. The value is the same as that obtained by the conventional boundary sedimentation method in the absence of the substrate. This result testifies to the fact that the conformation of reacting factor F1 in solution is similar to that of the enzyme in the absence of the substrate.