Planas Ferran, McLeish Michael J, Himo Fahmi
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-10691 Stockholm, Sweden.
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis IN 46202, USA.
Beilstein J Org Chem. 2019 Jan 16;15:145-159. doi: 10.3762/bjoc.15.15. eCollection 2019.
Thiamin diphosphate (ThDP)-dependent enzymes constitute a large class of enzymes that catalyze a diverse range of reactions. Many are involved in stereospecific carbon-carbon bond formation and, consequently, have found increasing interest and utility as chiral catalysts in various biocatalytic applications. All ThDP-catalyzed reactions require the reaction of the ThDP ylide (the activated state of the cofactor) with the substrate. Given that the cofactor can adopt up to seven states on an enzyme, identifying the factors affecting the stability of the pre-reactant states is important for the overall understanding of the kinetics and mechanism of the individual reactions. In this paper we use density functional theory calculations to systematically study the different cofactor states in terms of energies and geometries. Benzoylformate decarboxylase (BFDC), which is a well characterized chiral catalyst, serves as the prototypical ThDP-dependent enzyme. A model of the active site was constructed on the basis of available crystal structures, and the cofactor states were characterized in the presence of three different ligands (crystallographic water, benzoylformate as substrate, and ()-mandelate as inhibitor). Overall, the calculations reveal that the relative stabilities of the cofactor states are greatly affected by the presence and identity of the bound ligands. A surprising finding is that benzoylformate binding, while favoring ylide formation, provided even greater stabilization to a catalytically inactive tricyclic state. Conversely, the inhibitor binding greatly destabilized the ylide formation. Together, these observations have significant implications for the reaction kinetics of the ThDP-dependent enzymes, and, potentially, for the use of unnatural substrates in such reactions.
硫胺素二磷酸（ThDP）依赖性酶构成了一大类催化各种反应的酶。许多酶参与立体特异性碳-碳键的形成，因此，作为手性催化剂在各种生物催化应用中受到越来越多的关注和应用。所有ThDP催化的反应都需要ThDP叶立德（辅因子的活化状态）与底物反应。鉴于辅因子在酶上可以呈现多达七种状态，确定影响反应前状态稳定性的因素对于全面理解各个反应的动力学和机制至关重要。在本文中，我们使用密度泛函理论计算系统地研究了不同辅因子状态的能量和几何结构。苯甲酰甲酸脱羧酶（BFDC）是一种特征明确的手性催化剂，作为典型的ThDP依赖性酶。基于现有的晶体结构构建了活性位点模型，并在存在三种不同配体（结晶水、作为底物的苯甲酰甲酸和作为抑制剂的（）-扁桃酸）的情况下对辅因子状态进行了表征。总体而言，计算结果表明，辅因子状态的相对稳定性受结合配体的存在和性质的极大影响。一个惊人的发现是，苯甲酰甲酸的结合虽然有利于叶立德的形成，但对催化无活性的三环状态提供了更大的稳定性。相反，抑制剂的结合极大地破坏了叶立德的形成。这些观察结果共同对ThDP依赖性酶的反应动力学以及此类反应中使用非天然底物具有重要意义。
