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
Jm. Jault et al., 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, Biochemistry, 33(50), 1994, pp. 14979-14985
Lineweaver-Burk plots for ATP hydrolysis catalyzed by bovine heart mit
ochondrial 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), whic
h derivatizes noncatalytic nucleotide binding sites, is about 4 times
faster when loss of activity is monitored at 15 degrees C as opposed t
o 30 degrees C, This suggests that maximal loss of ATPase monitored at
15 degrees C is observed when a single noncatalytic site is derivatiz
ed, whereas maximal inactivation at 30 degrees C requires modification
of three noncatalytic sites. Prior incubation of MF(1) depleted of en
dogenous nucleotides (nd-MF(1)) with pyrophosphate (PPi) stimulates AT
Pase activity 2-fold when assayed at 30 degrees C and pH 8.0. This sti
mulation 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) i
ncreases 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 inactivat
ion induced by PPi when assays are conducted at 30 degrees C is not du
e 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 o
ne 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 catal
ytic sites by about 35%. Moreover, the range of concentration of PPi w
hich prevents labeling of noncatalytic sites by 2-N-3-[8-H-3]ADP is si
milar to the range that stimulates ATPase activity. These results firm
ly support the contention that PPi stimulates the ATPase activity of n
d-MF(1) by binding to noncatalytic sites which in turn promotes dissoc
iation of inhibitory MgADP from a catalytic site. The variations in th
e kinetic properties of ATP hydrolysis noted at 15 and 30 degrees C ra
ise 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 durin
g turnover at temperatures above 18 degrees C.