Active/inactive state transitions of the chloroplast F1 ATPase are induced by a slow binding and release of Mg2+. Relationship to catalysis and control of F1 ATPases.
Mg2+ is known to be a potent inhibitor of F1 ATPases from various sources. Such inhibition requires the presence of a tightly bound ADP at a catalytic site. Results with the spinach chloroplast F1 ATPase (CF1) show that the time delays of up to 1 min or more in the induction or the relief of the inhibition are best explained by a slow binding and slow release of Mg2+ rather than by slow enzyme conformational changes. CF1 is known to have multiple Mg2+ binding sites with Kd values in the micromolar range. The inhibitory Mg2+ and ADP can bind independently to CF1. When Mg2+ and ATP are added to the uninhibited enzyme, a relatively fast rate of hydrolysis attained soon after the addition is followed by a much slower steady-state rate. The inhibited steady-state rate results from a slowly attained equilibrium of binding of medium Mg2+. The Kd for the binding of the inhibitory Mg2+ is in the range of 1-8 microM, in the presence or absence of added ATP, as based on the extent of rate inhibition induced by Mg2+. Assessments from 18O exchange experiments show that the binding of Mg2+ is accompanied by a relatively rapid change to an enzyme form that is incapable of hydrolyzing MgATP. When ATP is added to the Mg2+- and ADP-inhibited enzyme, the resulting reactivation can be explained by MgATP binding to an alternate catalytic site which results in a displacement of the tightly bound ADP after a slow release of Mg2+. Both an increase in temperature (to 50 degrees C) and the presence of activating anions such as bicarbonate or sulfite reduce the extent of the Mg2+ inhibition markedly. The activating anions may bind to CF1 in place of Pi near the ADP. Whether the inhibitory Mg2+ binds at catalytic or noncatalytic nucleotide binding sites or at another location is not known. The Mg2(+)- and ADP-induced inhibition appears to be a general property of F1 ATPases, which show considerable differences in affinity for ADP, Mg2+, and Pi. These differences may reflect physiological control functions.
Guerrero
K J
KJ
Molecular Biology Institute, University of California, Los Angeles 90024.
Xue
Z X
ZX
Boyer
P D
PD
D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors*; D002736:Chloroplasts / Q000201:enzymology*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology*; D010944:Plants / Q000201:enzymology*; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013816:Thermodynamics
D006153:Guanosine Diphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases; D008274:Magnesium