Characteristics of the combination of inhibitory Mg2+ and azide with the F1 ATPase from chloroplasts.
The interactions between ADP, Mg2+, and azide that result in the inhibition of the chloroplast F1 ATPase (CF1) have been explored further. The binding of the inhibitory Mg2+ with low Kd is shown to occur only when tightly bound ADP is present at a catalytic site. Either the tightly bound ADP forms part of the Mg(2+)-binding site or it induces conformational changes creating the high-affinity site for inhibitory Mg2+. Kinetic studies show that CF1 forms two catalytically inactive complexes with Mg2+. The first complex results from Mg2+ binding with a Kd for Mg2+ dissociation of about 10-15 microM, followed by a slow conversion to a complex with a Kd of about 4 microM. The rate-limiting step of the CF1 inactivation by Mg2+ is the initial Mg2+ binding. When medium Mg2+ is chelated with EDTA, the two complexes dissociate with half-times of about 1 and 7 min, respectively. Azide enhances the extent of Mg(2+)-dependent inactivation by increasing the affinity of the enzyme for Mg2+ 3-4 times and prevents the reactivation of both complexes of CF1 with ADP and Mg2+. This results from decreasing the rate of Mg2+ release; neither the rate of Mg2+ binding to CF1 nor the rate of isomerization of the first inactive complex to the more stable form is affected by azide. This suggests that the tight-binding site for the inhibitory azide requires prior binding of both ADP and Mg2+.
Murataliev
M B
MB
Molecular Biology Institute, University of California, Los Angeles 90024-1570.
Milgrom
Y M
YM
Boyer
P D
PD
D001386:Azides / Q000494:pharmacology*; D002384:Catalysis; D002736:Chloroplasts / Q000187:drug effects / Q000201:enzymology*; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D008274:Magnesium / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000187:drug effects
D001386:Azides; D006180:Proton-Translocating ATPases; D008274:Magnesium