LOWERED TEMPERATURE OR BINDING OF PYROPHOSPHATE TO SITES FOR NONCATALYTIC NUCLEOTIDES MODULATES THE ATPASE ACTIVITY OF THE BEEF-HEART MITOCHONDRIAL F1-ATPASE BY DECREASING THE AFFINITY OF A CATALYTIC SITE FOR INHIBITORY MGADP

Lineweaver-Burk plots for ATP hydrolysis catalyzed by bovine heart mitochondrial F-1-ATPase (MFI) at 30 degrees C are biphasic, whereas they are linear at 15 degrees C. The rate of inactivation of the enzyme at 23 degrees C by 5'-[(p-fluorosulfonyl)benzoyl] adenosine (FSBA), which derivatizes noncatalytic nucleotide binding sites, is about 4 times faster when loss of activity is monitored at 15 degrees C as opposed to 30 degrees C, This suggests that maximal loss of ATPase monitored at 15 degrees C is observed when a single noncatalytic site is derivatized, whereas maximal inactivation at 30 degrees C requires modification of three noncatalytic sites. Prior incubation of MF(1) depleted of endogenous nucleotides (nd-MF(1)) with pyrophosphate (PPi) stimulates ATPase activity 2-fold when assayed at 30 degrees C and pH 8.0. This stimulation correlates with binding of [P-32]PPi to the second and third binding sites for PPi to be filled. Prior binding of PPi to nd-MF(1) increases the rate of inactivation of the enzyme by FSBA at 23 degrees C about 4-fold when loss of activity is monitored at 30 degrees C and pH 8.0, whereas it does not affect the rate of inactivation when loss of ATPase is monitored at 15 degrees C or loss of ITPase is monitored at 30 degrees C. This indicates that the accelerated rate of inactivation induced by PPi when assays are conducted at 30 degrees C is not due to an increased rate of derivatization of noncatalytic sites. After 85% inactivation with FSBA, nd-MF(1) retains the capacity to bind 2.8 mol of [P-32]PPi per mole. However, on modification of approximately one noncatalytic site with FSBA, the stimulatory effect induced by PPi is lost. Prior incubation of nd-MF(1) with PPi eliminates labeling of noncatalytic sites with 2-N-3-[H-3]ADP and decreases labeling of catalytic sites by about 35%. Moreover, the range of concentration of PPi which prevents labeling of noncatalytic sites by 2-N-3-[8-H-3]ADP is similar to the range that stimulates ATPase activity. These results firmly support the contention that PPi stimulates the ATPase activity of nd-MF(1) by binding to noncatalytic sites which in turn promotes dissociation of inhibitory MgADP from a catalytic site. The variations in the kinetic properties of ATP hydrolysis noted at 15 and 30 degrees C raise the possibility that the break in the Arrhenius plot at 18 degrees C for ATP hydrolysis originally described by Harris et al. [Harris, D. A., et al. (1981) Biochim. Biophys. Acta 635, 412-428] is caused by entrapment of inhibitory MgADP in a catalytic site of the enzyme during turnover at temperatures above 18 degrees C.