Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase.

H S Penefsky

doi:10.1016/s0021-9258(17)38787-2

Abstract

Incubation of [gamma-32P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma-32P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma-32P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide.

Highlights

Subsequentinitiationof respiration by additiofnsuc- of other charged amino acid residues in Fo
If the high affinity catalytic sites of membrane-bound F1participate inATP synthesis inoxidative phosphorylation, it may expected that these siteswill exhibit energy-dependent changes in affinity for bound ATP
The steady state rate of respiration catalyzed by the actisupport a model for ATP synthesis inoxidative phosphorylation which proposes that ATPforms from ADP and Pibound in high affinity catalytic siteswith little or no change in free energy and that thmeajor requirement for energy in oxidative vated submitochondrial particles was 0.25pA of O/min/mg of protein with succinate as substrate and 0.4-0.5pAof O/min/mg of protein with NADH

Summary

RESULTS

5 s after addition inhibitors described in this paper, ethanol alone (line d) was of [y-32P]ATP,NADH was added and theincubation was continued without effect on NADH-dependent dissociation of [r-”P]. The reaction mixtures contained, in a final volume of 1 ml, 20 mM Tris-SO,, pH 8, 5 mM MgS04, 1 mgof activated submitochondrial particles (specific activity = 9 units/mg), 0.12 nmol of [3H,y-32P]ATP and, where shown, 4 mM Pi and 1 mM NADH. A total of 33% of the bound I radioactive nucleotide is dissociated after about 3 s of incu-

NO NADH

DISCUSSION

With NADH NADH