Atomistic details of the Catalytic Mechanism of Fe(III)-Zn(II) Purple Acid Phosphatase
Atomistic details of the Catalytic Mechanism of Fe(III)-Zn(II) Purple Acid Phosphatase
复制标题
DOI：
10.1021/ct100187c
复制
发表时间：
2010-08-01
影响因子：
5.5
通讯作者：
Russo, Nino
中科院分区：
文献类型：
--
作者：
Alberto, Marta E.;Marino, Tiziana;Russo, Nino
本文利用杂交密度泛函理论和不同的交换相关电位，对红芸豆中Fe(III)-Zn（II）紫色酸性磷酸酶（rkbPAP）的反应机理进行了理论研究。报道了不同环境（气相、蛋白质环境和水）下反应的过渡态和中间体的表征以及势能分布。我们的研究结果表明，Fe(III)-Zn(II)PAP通过桥接金属配位氢氧化物的直接攻击催化甲基磷酸盐的水解，导致酯键的断裂。我们的研究表明，该反应的限速步骤是亲核攻击，然后是能量要求较低的离去基的释放。我们提供了一些关于预催化配合物和底物配合模式进入水解前活性位点的重要争论点的见解。(i)测试了两种不同底物进入活性位点方向的酶-底物模型，以评估保守组氨酸残基（His 202和His 296）可能发挥的作用。（ii）考虑了底物的不同质子化状态，以重现不同的pH值，并验证其对催化效率和底物结合模式的影响；（三）阐明了金属在催化机制各步骤中的作用。我们还能够确定，质子化的His 296对离去基的活化是达到最佳催化效率的决定性因素，而没有活化的键断裂需要更高的能量才能发生。
In the present work, we performed a theoretical investigation of the reaction mechanism of the Fe(III)-Zn(II) purple acid phosphatase from red kidney beans (rkbPAP), using the hybrid density functional theory and employing different exchange-correlation potentials. Characterization of the transition states and intermediates involved and the potential energy profiles for the reaction in different environments (gas phase, protein environment, and water) are reported. Our results show that the Fe(III)-Zn(II)PAP catalyzes the hydrolysis of methylphosphate via direct attack by a bridging metals-coordinated hydroxide leading to the cleavage of the ester bond From our study emerges that the rate-limiting step of the reaction is the nucleophilic attack followed by the less energetically demanding release of the leaving group Furthermore, we provide insights into some important points of contention concerning the precatalytic complex and the substrate coordination mode into the active site prior to hydrolysis In particular. (i) Two models of enzyme-substrate with different orientations of the substrate into the active site were tested to evaluate the possible roles played by the conserved histidine residues (His 202 and His 296). (ii) Different protonation states of the substrate were taken into account in order to reproduce different pH values and to verify its influence on the catalytic efficiency and on the substrate binding mode; (iii) The metals role in each step of the catalytic mechanism was elucidated. We were also able to ascertain that the activation of the leaving group by the protonated His 296 is decisive to reach an optimal catalytic efficiency, while the bond scission without activation requires higher energy to occur.