Institute of Bioorganic Chemistry Russian Academy of Sciences, Moscow 117997, Russia.
Proc Natl Acad Sci U S A. 2011 Sep 20;108(38):15954-9. doi: 10.1073/pnas.1108460108. Epub 2011 Sep 6.
Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (V(L)) and variable heavy (V(H)) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.
Igs 为从头设计具有催化活性的蛋白质提供了一种多功能模板，适用于组合和合理的设计方法。我们使用共价捕获选择策略，从使用亲核机制的人半合成抗体可变片段文库中鉴定出生物催化剂。在使用亲核机制的人半合成抗体可变片段文库中鉴定出生物催化剂。在重组 IgG 构建体中确认了在可变轻链框架内的单个酪氨酸的特异性膦酸化。未修饰和膦酸化 Fab 的高分辨率晶体结构显示在可变轻链（V(L)）和可变重链（V(H)）片段的界面处存在一个 15Å 深的双腔室，在该位置的底部具有亲核酪氨酸。口袋的深度和结构通常与抗体不同，但可以与胆碱酯酶的催化位点进行定性比较。结构上无序的重链互补决定区 3 环，构成裂缝的一堵墙，在共价修饰后通过与膦酸三醇部分形成氢键而稳定。这些特征和预稳态动力学分析表明，该反应中存在诱导契合机制。位于该稳定环中的残基的突变不会干扰与有机磷配体的直接接触，但可以检验第二壳层相互作用，因为 H3 环具有适合结合的构象。动力学和热力学参数以及计算对接支持活性位点模型，包括可塑性和简单的催化组件。尽管相对简单，但这种催化机制既显示出立体选择性，又显示出化学选择性。有机磷农药对氧磷通过共价催化进行水解，限速步骤为去磷酸化。因此，这种反应抗体是一种具有酶样催化属性的动力学选择的蛋白质模板。
