ACCELERATION OF UNISITE CATALYSIS OF MITOCHONDRIAL F-1-ADENOSINE-TRIPHOSPHATASE BY ATP, ADP AND PYROPHOSPHATE - HYDROLYSIS AND RELEASE OF THE PREVIOUSLY BOUND [GAMMA-P-32]ATP

The effect of ATP, ADP and pyrophosphate (PPi) on hydrolysis and relea se of [gamma-P-32]ATP bound to the high-affinity catalytic site of sol uble F-1 from bovine heart mitochondria under unisite conditions [Grub meyer, C., Cross, R. L. & Penefsky, H. S. (1982) J. Biol. Chem. 257, 1 2092-12100] was studied. In accord with the previous data, it was obse rved that millimolar concentrations of ATP or ADP added to F-1 undergo ing unisite hydrolysis of [gamma-P-32]ATP accelerated its hydrolysis. PPi also produced a hydrolytic burst of a fraction of the previously b ound [gamma-P-32]ATP; kinetic data suggested that for production of op timal hydrolysis by PPi of the bound [gamma-P-32]ATP, two binding site s with apparent K-d of 27 mu M and 240 mu M must be filled. The extent of the hydrolytic burst induced by MgPPi was lower than that induced by ADP and ATP. In F-1 in which PPi had produced a hydrolytic burst of the bound [gamma-P-32]ATP, the addition of ATP induced a second burst of hydrolysis. By filtration experiments and enzyme trapping, it was also studied whether ATP, ADP and PPi produce release of the tightly b ound [gamma-P-32]ATP. At millimolar concentrations. ATP and ADP brough t about release of about 25 % of the previously bound [gamma-P-32]ATP. At micromolar concentrations, ADP accelerated the hydrolysis of the p reviously bound [gamma-P-32]ATP but not its release. Hence, the hydrol ytic and release reactions could be separated, indicating that the two reactions require the occupancy of different sites in F-1. With PPi, no release of the tightly bound [gamma-P-32]ATP was observed. The ADP induced hydrolysis and release of the F-1-bound [gamma-P-32]ATP were i nhibited by sodium azide to the same extent (60%). Since release of AT P from a high-affinity calalytic site of F-1 represents the terminal s tep of oxidative phosphorylation. the data illustrate that the binding energy of substrates to F-1 is critical to the ejection of ATP into t he media. The failure of PPi to induce release of [gamma-P-32]ATP boun d to F-1 under unisite conditions is probably due to its lower binding energy.