Sawicki Artur, Willows Robert D
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.
J Biol Chem. 2008 Nov 14;283(46):31294-302. doi: 10.1074/jbc.M805792200. Epub 2008 Sep 12.
The metabolic pathway known as (bacterio)chlorophyll biosynthesis is initiated by magnesium chelatase (BchI, BchD, BchH). This first step involves insertion of magnesium into protoporphyrin IX (proto), a process requiring ATP hydrolysis. Structural information shows that the BchI and BchD subunits form a double hexameric enzyme complex, whereas BchH binds proto and can be purified as BchH-proto. We utilized the Rhodobacter capsulatus magnesium chelatase subunits using continuous magnesium chelatase assays and treated the BchD subunit as the enzyme with both BchI and BchH-proto as substrates. Michaelis-Menten kinetics was observed with the BchI subunit, whereas the BchH subunit exhibited sigmoidal kinetics (Hill coefficient of 1.85). The BchI.BchD complex had intrinsic ATPase activity, and addition of BchH greatly increased ATPase activity. This was concentration-dependent and gave sigmoidal kinetics, indicating there is more than one binding site for the BchH subunit on the BchI.BchD complex. ATPase activity was approximately 40-fold higher than magnesium chelatase activity and continued despite cessation of magnesium chelation, implying one or more secondary roles for ATP hydrolysis and possibly an as yet unknown switch required to terminate ATPase activity. One of the secondary roles for BchH-stimulated ATP hydrolysis by a BchI.BchD complex is priming of BchH to facilitate correct binding of proto to BchH in a form capable of participating in magnesium chelation. This porphyrin binding is the rate-limiting step in catalysis. These data suggest that ATP hydrolysis by the BchI.BchD complex causes a series of conformational changes in BchH to effect substrate binding, magnesium chelation, and product release.
被称为（细菌）叶绿素生物合成的代谢途径由镁螯合酶（BchI、BchD、BchH）启动。第一步涉及将镁插入原卟啉IX（原卟啉），这一过程需要ATP水解。结构信息表明，BchI和BchD亚基形成一个双六聚体酶复合物，而BchH结合原卟啉，并可以以BchH-原卟啉的形式纯化。我们利用荚膜红细菌镁螯合酶亚基进行连续的镁螯合酶测定，并将BchD亚基视为酶，以BchI和BchH-原卟啉作为底物。BchI亚基呈现米氏动力学，而BchH亚基呈现S形动力学（希尔系数为1.85）。BchI.BchD复合物具有内在的ATP酶活性，添加BchH可大大增加ATP酶活性。这是浓度依赖性的，并呈现S形动力学，表明BchH亚基在BchI.BchD复合物上有多个结合位点。ATP酶活性比镁螯合酶活性高约40倍，并且尽管镁螯合停止仍继续，这意味着ATP水解有一个或多个次要作用，可能还有一个尚未知的终止ATP酶活性所需的开关。BchH刺激BchI.BchD复合物进行ATP水解的一个次要作用是使BchH引发，以促进原卟啉以能够参与镁螯合的形式正确结合到BchH上。这种卟啉结合是催化中的限速步骤。这些数据表明，BchI.BchD复合物进行的ATP水解会导致BchH发生一系列构象变化，以实现底物结合、镁螯合和产物释放。
