Mechanism of 4-chlorobenzoate: Coenzyme a ligase catalysis
Mechanism of 4-chlorobenzoate: Coenzyme a ligase catalysis
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DOI：
10.1021/bi800698m
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发表时间：
2008-08-05
期刊：
影响因子：
2.9
通讯作者：
Dunaway-Mariano, Debra
中科院分区：
文献类型：
--
作者：
Wu, Rui;Cao, Jian;Dunaway-Mariano, Debra
在本期的随附论文中（Reger等人（2008）Biochemistry，47，8016-8025），我们报道了与4-氯苯甲酰基腺苷酸（4-CB-AMP）结合的4-氯苯甲酸：CoA连接酶（CBL）的X射线结构和与4-氯苯甲酰辅酶A（4-CP-CoA）（产物4-氯苯甲酰基辅酶A（4-CB-CoA）的惰性类似物）和AMP结合的CBL的X射线结构。这些结构定义了两种CBL构象状态。在构象1中，CBL准备催化4-氯苯甲酸（4-CB）与ATP的腺苷酸化（部分反应1），在构象2中，CBL准备催化4-CB-AMP和CoA形成4-CB-CoA（部分反应2）。这两种结构表明，通过从构象I转换为构象2，帽结构域围绕结构域接头旋转，从而改变其与N-末端结构域的界面。目前的工作进行，以确定每个活性位点残基在底物/辅因子结合和催化的贡献，并测试结构域的交替在催化中的作用。在本文中，我们报告的结果，稳态动力学和瞬态动力学分析的野生型CBL和一系列的定点CBL活性位点突变体。主要研究结果如下。首先，野生型CBL被Mg 2+激活（根据测定条件观察到活性增加12-75倍），其动力学机制（乒乓）支持结构衍生的预测，即PPi解离必须先于从构象1转换为构象2，因此CoA结合。此外，野生型CBL的瞬时动力学分析确定了催化反应的限速步骤为4-CB-CoA形成后的步骤（即CBL构象变化和/或产物解离）。4-CB和ATP形成4-CB-AMP和PPi的单一周转率（k = 300 s（-1））不受CoA存在的影响，并且其类似于比4-CB-AMP和CoA形成4-CB-CoA和AMP的周转率（k = 120 s（-1））快3倍。其次，基于在任何一个底物k（cat）/Km值中观察到的降低程度，对通过稳态动力学分析筛选的活性位点突变体进行排序，并且选择kcat/Km值降低50倍以上的那些得分用于通过瞬态动力学分析进行进一步评价。的单周转时间过程中，测量的第一部分反应，然后为整个反应，进行了分析，以确定微观速率常数的腺苷酸化反应和硫酯化反应。我们的研究结果的基础上，我们提出了一个催化机制，集中在一小群关键残基（其中一些服务于一个以上的角色），包括几个残基，功能域交替。
Within the accompanying paper in this issue (Reger et al. (2008) Biochemistry, 47, 8016-8025) we reported the X-ray structure of 4-chlorobenzoate:CoA ligase (CBL) bound with 4-chlorobenzoyladenylate (4-CB-AMP) and the X-ray structure of CBL bound with 4-chlorophenacyl-CoA (4-CP-CoA) (an inert analogue of the product 4-chlorobenzoyl-coenzyme A (4-CB-CoA)) and AMP. These structures defined two CBL conformational states. In conformation 1, CBL is poised to catalyze the adenylation of 4-chlorobenzoate (4-CB) with ATP (partial reaction 1), and in conformation 2, CBL is poised to catalyze the formation of 4-CB-CoA from 4-CB-AMP and CoA (partial reaction 2). These two structures showed that, by switching from conformation I to conformation 2, the cap domain rotates about the domain linker and thereby changes its interface with the N-terminal domain. The present work was carried out to determine the contributions made by each of the active site residues in substrate/cofactor binding and catalysis, and also to test the role of domain alternation in catalysis. In this paper, we report the results of steady-state kinetic and transient state kinetic analysis of wild-type CBL and of a series of site-directed CBL active site mutants. The major findings are as follows. First, wild-type CBL is activated by Mg2+ (a 12-75-fold increase in activity is observed depending on assay conditions) and its kinetic mechanism (ping-pong) supports the structure-derived prediction that PPi dissociation must precede the switch from conformation 1 to conformation 2 and therefore CoA binding. Also, transient kinetic analysis of wildtype CBL identified the rate-limiting step of the catalyzed reaction as one that follows the formation of 4-CB-CoA (viz. CBL conformational change and/or product dissociation). The single turnover rate of 4-CB and ATP to form 4-CB-AMP and PPi (k = 300 s(-1)) is not affected by the presence of CoA, and it is similar to 3-fold faster than the turnover rate of 4-CB-AMP and CoA to form 4-CB-CoA and AMP (k = 120 s(-1)). Second, the active site mutants screened via steady-state kinetic analysis were ranked based on the degree of reduction observed in any one of the substrate k(cat)/K-m values, and those scoring higher than a 50-fold reduction in kcat/Km value were selected for further evaluation via transient state kinetic analysis. The single-turnover time courses, measured for the first partial reaction, and then for the full reaction, were analyzed to define the microscopic rate constants for the adenylation reaction and the thioesterification reaction. On the basis of our findings we propose a catalytic mechanism that centers on a small group of key residues (some of which serve in more than one role) and that includes several residues that function in domain alternation.