Tight divalent metal binding to Escherichia coli F1-adenosinetriphosphatase. Complete substitution of intrinsic magnesium by manganese or cobalt and studies of metal binding sites.
Tight divalent metal binding sites in Escherichia coli F1-adenosinetriphosphatase (F1-ATPase) were studied. Native enzyme contained two Mg per F1, confirming previous results. All of the Mg may be replaced by Co or Mn using a dissociation-repolymerization procedure. The substituted enzymes are homogeneous and contain two Mn per F1 or two Co per F1. They are fully active as ATPases, they rebind to F1-depleted membranes, and they catalyze ATP-driven proton pumping. N,N'-Dicyclohexylcarbodiimide-(DCCD) inactivated F1 retains all the intrinsic tightly bound Mg. Evidence is presented that DCCD affects at least two beta subunits in E. coli F1, and therefore, the tightly bound metals appear not to be bound at the DCCD-reactive glutamate residue on the beta subunit. However, the nature of the tightly bound metal (Mg, Mn, or Co) as well as the presence of added (2 mM) MgSO4, MnSO4, or CoSO4 affected the rate of DCCD inactivation, showing that metal binding changes the beta-subunit conformation. Isolated F1 alpha subunit bound Mg, Mn, or Co stoichiometrically and independently of ATP binding. Isolated F1 beta subunit bound only small amounts of Mg, and no Co or Mn. Therefore, it is possible, although not conclusively shown, that the alpha subunit is the site of tight metal binding in the intact F1.
Smith
R A
RA
Latchney
L R
LR
Senior
A E
AE
D001665:Binding Sites; D003035:Cobalt / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D046911:Macromolecular Substances; D003035:Cobalt; D008345:Manganese; D004024:Dicyclohexylcarbodiimide; D006180:Proton-Translocating ATPases; D008274:Magnesium