CHARACTERISTICS OF THE COMBINATION OF INHIBITORY MG2+ AND AZIDE WITH THE F1 ATPASE FROM CHLOROPLASTS
CHARACTERISTICS OF THE COMBINATION OF INHIBITORY MG2+ AND AZIDE WITH THE F1 ATPASE FROM CHLOROPLASTS
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DOI：
10.1021/bi00098a004
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发表时间：
1991-08-27
期刊：
影响因子：
2.9
通讯作者：
BOYER, PD
中科院分区：
文献类型：
--
作者：
MURATALIEV, MB;MILGROM, YM;BOYER, PD
进一步探讨了ADP、Mg2+和叠氮化物之间的相互作用导致叶绿体F1-ATPase(CF1)的抑制。只有当紧密结合的ADP存在于催化部位时，抑制镁离子才能与低K(D)发生结合。要么是紧密结合的ADP形成了镁离子结合位点的一部分，要么是它诱导了构象变化，为抑制性镁离子创造了高亲和力的位点。动力学研究表明，CF1与镁离子形成两种催化失活的络合物。第一个络合物是由镁离子与K(D)结合形成的，其解离约为10-15-mU-M，然后缓慢转化为K(D)约为4-mU-M的络合物。镁离子使CF1失活的限速步骤是初始的镁离子结合。当介质中的镁离子与EDTA络合时，两种络合物的解离半衰期分别约为1和7min。叠氮可使酶对镁离子的亲和力增加3-4倍，从而增强对镁离子依赖的失活程度，并阻止CF1与ADP和镁离子的两种络合物的重新激活。这是由于降低了镁离子的释放速度；叠氮化物既不影响镁离子与CF1的结合速度，也不影响第一个失活的络合物异构化为更稳定的形式的速度。这表明抑制叠氮化物的紧密结合部位需要ADP和Mg2+的预先结合。
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 K(d) is shown to occur only when tightly bound ADP is present at a catalytic site. Either the tightly bound ADP forms part of the Mg2+-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 K(d) for Mg2+ dissociation of about 10-15-mu-M, followed by a slow conversion to a complex with a K(d) of about 4-mu-M. 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 Mg2+-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+.