Kinetic and microcalorimetric analysis of substrate and cofactor interactions in epoxyalkane:CoM transferase, a zinc-dependent epoxidase.
Kinetic and microcalorimetric analysis of substrate and cofactor interactions in epoxyalkane:CoM transferase, a zinc-dependent epoxidase.
复制标题
环氧烷烃：CoM 转移酶（一种锌依赖性环氧酶）中底物和辅因子相互作用的动力学和微量热分析。
DOI：
10.1021/bi0255221
复制
发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Ensign,ScottA
中科院分区：
文献类型：
--
作者：
Krum,JonathanG;Ellsworth,Heather;Sargeant,RyanR;Rich,Gregory;Ensign,ScottA
环氧烷烃：辅酶M转移酶（Epoxyalkane：CoM transferase，EaCoMT）是细菌丙烯代谢的关键酶，催化辅酶M（CoM，2-巯基乙磺酸）亲核攻击环氧丙烷，生成硫醚共轭物2-羟丙基-CoM。EaCoMT的生物化学和分子特性表明，该酶属于烷基转移酶家族，其中Zn在激活有机硫醇底物对烷基供体底物进行亲核攻击中起关键作用。在目前的工作中，锌在EaCoMT催化的反应中的作用是通过从EaCoMT中去除锌，导致在添加锌后恢复的酶的催化活性的损失，并通过表达通过添加ZnCl 2或CoCl 2激活的酶的无活性和锌缺乏的形式来建立的。预测的锌配体之一（C220 A）的定点突变导致形成了一个很大程度上无催化活性的蛋白质（野生型活性的0.06%），当纯化时，含有锌的亚化学计量的补充。对EaCoMT进行了动力学表征，发现其遵循随机序列机理，动力学参数Km，环氧丙烷= 1.8 μM，Km，CoM= 34 μM，kcat = 6.5 s-1。CoM类似物2-巯基丙酸酯，2-巯基乙醇，和半胱氨酸取代CoM作为巯基底物差，环氧烷烃共轭的具体速率最多为0.6%的CoM依赖率，而乙硫醇，丙硫醇，谷胱甘肽，同型半胱氨酸，和硫辛酸提供没有活性。2-巯基乙醇是一个弱的竞争性抑制剂与CoM与aKI的192 mM。等温滴定量热法被用来研究CoM和类似物与holo，Zn-缺陷，和C220 A EaCoMT变体的相互作用的热力学结合决定因素。CoM结合的化学计量与Zn含量而不是蛋白质样品的单体含量直接相关，加强了Zn在CoM结合中的重要性。CoM与EaCoMT的结合发生在ΔG= −7.5 kcal/mol（Kd= 3.8 μM），并由大量焓释放驱动。测定了CoM、乙磺酸盐和乙硫醚单独结合的热力学贡献因子（Ka、ΔG、ΔH、ΔS），并用于评估硫醇、烷基和磺酸盐部分对E·CoM二元复合物中总结合能的贡献。
Epoxyalkane:CoM transferase (EaCoMT) is a key enzyme of bacterial propylene metabolism, catalyzing the nucleophilic attack of coenzyme M (CoM, 2-mercaptoethanesulfonic acid) on epoxypropane to form the thioether conjugate 2-hydroxypropyl-CoM. The biochemical and molecular properties of EaCoMT suggest that the enzyme belongs to the family of alkyltransferase enzymes for which Zn plays a key role in activating an organic thiol substrate for nucleophilic attack on an alkyl-donating substrate. In the present work, the role of Zn in the EaCoMT-catalyzed reactions is established by removing Zn from EaCoMT, resulting in loss of catalytic activity that was restored upon addition of Zn back to the enzyme, and by expressing an inactive and Zn-deficient form of the enzyme that was activated by addition of ZnCl2or CoCl2. Site-directed mutagenesis of one of the predicted Zn ligands (C220A) resulted in the formation of a largely catalytically inactive protein (0.06% of wild-type activity) that, when purified, contained a substoichiometric complement of Zn. EaCoMT was kinetically characterized and found to follow a random sequential mechanism with kinetic parametersKm,epoxypropane= 1.8 μM,Km,CoM= 34 μM, andkcat= 6.5 s-1. The CoM analogues 2-mercaptopropionate, 2-mercaptoethanol, and cysteine substituted poorly for CoM as the thiol substrate, with specific rates of epoxyalkane conjugation that were at best 0.6% of the CoM-dependent rate, while ethanethiol, propanethiol, glutathione, homocysteine, and lipoic acid provided no activity. 2-Mercaptoethanol was a weak competitive inhibitor vs CoM with aKIof 192 mM. Isothermal titration calorimetry was used to investigate the thermodynamic binding determinants for the interaction of CoM and analogues with holo, Zn-deficient, and C220A EaCoMT variants. The stoichiometry of CoM binding correlated directly with the Zn content rather than monomer content of protein samples, reinforcing the importance of Zn in CoM binding. The binding of CoM to EaCoMT occurred with ΔG= −7.5 kcal/mol (Kd= 3.8 μM) and was driven by a large release of enthalpy. The thermodynamic contributors (Ka, ΔG, ΔH, ΔS) to the individual binding of CoM, ethanesulfonate, and ethanethiol were determined and used to assess the contributions of the thiol, alkyl, and sulfonate moieties to total binding energy in the E·CoM binary complex.