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 F1-ATPase (MF1) 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 MF1 depleted of endogenous nucleotides (nd-MF1) with pyrophosphate (PPi) stimulates ATPase activity 2-fold when assayed at 30 degrees C and pH 8.0. This stimulation correlates with binding of [32P]PPi to the second and third binding sites for PPi to be filled. Prior binding of PPi to nd-MF1 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-MF1 retains the capacity to bind 2.8 mol of [32P]PPi per mole.(ABSTRACT TRUNCATED AT 250 WORDS)
Jault
J M
JM
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla 92093-0601.
Paik
S R
SR
Grodsky
N B
NB
Allison
W S
WS
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D003080:Cold Temperature; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014316:Tritium
D009711:Nucleotides; D010710:Phosphates; D014316:Tritium; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases