Nucleotide binding sites on beef heart mitochondrial F1-ATPase. Cooperative interactions between sites and specificity of noncatalytic sites.
We have studied the properties of beef heart mitochondrial F1 having inhibitory MgADP bound at one of the three catalytic sites and various levels of occupancy of the three noncatalytic nucleotide sites including zero, two, or three ADP/ATPs or two ADP/ATP plus one GTP. The properties examined include the rate of MgATP-dependent reactivation and the rate of increase in the fraction of F1 containing transiently bound intermediates. For each form of the enzyme tested, the rate of reactivation closely paralleled the rate of increase in the level of bound intermediates, indicating that when one catalytic site on F1 is blocked by inhibitory MgADP, the remaining two sites are incapable of residual uni- or bi-site activity. It was also found that the stability of the MgADP-inhibited complex decreases with full occupancy of the noncatalytic sites. This demonstrates that the noncatalytic sites modulate the properties of catalytic sites. Finally, it was found that the noncatalytic sites on mitochondrial F1 do not, as has long been believed, bind adenine nucleotides exclusively. Evidence is presented that both GTP and PPi bind tightly at noncatalytic sites.
Milgrom
Y M
YM
Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse 13210.
Cross
R L
RL
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D011756:Diphosphates / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D008929:Mitochondria, Heart / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism*
D011756:Diphosphates; D012265:Ribonucleotides; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases