Direct measurement of metal-ion chelation in the active site of the AAA+ ATPase magnesium chelatase.
Direct measurement of metal-ion chelation in the active site of the AAA+ ATPase magnesium chelatase.
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直接测量 AAA ATPase 镁螯合酶活性位点的金属离子螯合。
DOI：
10.1021/bi701515y
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发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
J. D. Reid
中科院分区：
文献类型：
--
作者：
J. Viney;Paul A. Davison;C. Hunter;J. D. Reid
镁螯合酶催化叶绿素生物合成的第一个关键步骤。该复合酶具有至少三种底物，并将ATP水解与Mg 2+插入原卟啉IX中偶联。我们直接观察到金属离子螯合荧光，提供了第一个数据描述的酶反应。我们描述了镁螯合酶的瞬态动力学与直接观察的酶产物复合物EMgDIX的演变。我们证明，MgATP 2结合后发生的速率决定步骤。由于核苷酸水解对于整个反应是必不可少的，这也必须在速率决定步骤之后发生。这为ATP酶和螯合酶途径的同步化提供了第一个证据，并提出了一种机制，其中核苷酸结合作用将螯合酶夹在产物复合物中。在反应中的缓慢步骤的速率常数与进一步的瞬态动力学的条件下，可以发生多个周转的比较表明，需要一个额外的活化步骤来解释镁螯合酶的行为。这些数据提供了一个新的视图中发生的事件在镁螯合酶催化的反应的顺序。
Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis. This complex enzyme has at least three substrates and couples ATP hydrolysis to the insertion of Mg2+ into protoporphyrin IX. We directly observed metal-ion chelation fluorometrically, providing the first data describing the on-enzyme reaction. We describe the transient-state kinetics of magnesium chelatase with direct observation of the evolution of an enzyme-product complex EMgDIX. We demonstrate that MgATP2- binding occurs after the rate-determining step. As nucleotide hydrolysis is essential for the overall reaction this must also occur after the rate-determining step. This provides the first evidence for the synchronization of the ATPase and chelatase pathways and suggests a mechanism where nucleotide binding acts to clamp the chelatase in a product complex. Comparison of rate constants for the slow step in the reaction with further transient kinetics under conditions where multiple turnovers can occur reveals that an additional activation step is required to explain the behavior of magnesium chelatase. These data provide a new view of the sequence of events occurring in the reaction catalyzed by magnesium chelatase.