Influence of divalent cations on nucleotide exchange and ATPase activity of chloroplast coupling factor 1.
The ATPase activity of the catalytic part of ATP synthases is inhibited by free Mg2+, even though MgATP is the substrate. Here we show that the inhibition of the MgATPase activity of chloroplast coupling factor 1 deficient in its epsilon subunit (CF1-epsilon) by Mg2+ is complex. The hydrolysis of MgATP by CF1-epsilon that contains tightly bound ADP, but no bound Mg2+, is initially rapid and decreases within about 1 min to a steady-state rate. The bound MgADP content of CF1-epsilon was varied. The initial fast phase of MgATP hydrolysis is eliminated when the molar ratio of MgADP to CF1-epsilon approaches 2. Loosely bound Mg2+ also affects the initial kinetics of the enzyme that contains bound MgADP. At molar ratios of bound MgADP to enzyme in excess of 1, the initial ATPase activity was low and reached the steady state after about 30 s. Free Mg2+ in the assay mix also inhibited steady-state ATP hydrolysis by all forms of the enzyme. The results are consistent with a model in which two Mg2+ bind cooperatively, probably to the dissociable nucleotide-binding sites on CF1-epsilon. Thus, four different nucleotide-binding sites may be involved in the inhibition of the MgATPase activity of CF1-epsilon. Three of these sites are potentially catalytic, and the fourth may be regulatory. The exchange of bound trinitrophenyl-ADP induced by the addition of MgATP or CaATP was found to be fast enough for the site to be involved in catalysis.
Digel
J G
JG
The Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Moore
N D
ND
McCarty
R E
RE
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D004789:Enzyme Activation; D006868:Hydrolysis; D007700:Kinetics; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D018724:Spinacia oleracea; D013997:Time Factors
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases