Biomimetic catalysis of intermodular aminoacyl transfer
Biomimetic catalysis of intermodular aminoacyl transfer
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DOI：
10.1021/ja067124h
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发表时间：
2007-01-31
影响因子：
15
通讯作者：
Ghadiri, M. Reza
中科院分区：
文献类型：
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作者：
Wilcoxen, Keith M.;Leman, Luke J.;Ghadiri, M. Reza
模间氨基酰基转移是核糖体和非核糖体肽合成酶（NRPS）合成多肽过程中最基本的键形成反应。在这里，我们报告了短合成α -螺旋肽的设计和功能表征，这些肽模拟了NRPS的氨基酰负载和氨基酰间转移步骤，并在高达5400倍(k（cat)/k(uncat)）的中性水溶液中提高了氨基水解率。催化剂作为具有复合活性位点的非共价相关肽组件在螺旋亚基之间的界面形成。在活性位点半胱氨酸处装载底物后，所产生的氨基酰基硫酯和亲核胺的酰基受体片段并置于一起，产生了高有效浓度（高达54 M），促进了螺旋间氨基酰基转移，其速率通常超过10（-4）秒（-1）。此外，基于同源和异聚体组装、活性位点氨基酸取代、动力学分析和反应模型的研究表明，本文报道的新设计的超分子催化剂具有天然酶的一些基本特征，包括活性位点残基的精确定位和pK(a)调制、共价催化和多产物转换。
Intermodular aminoacyl transfer is the fundamental bond-forming reaction in the biosynthesis of polypeptides by ribosomes and nonribosomal peptide synthetases (NRPS). Here we report the design and functional characterizations of short synthetic alpha-helical peptides that mimic the aminoacyl loading and intermodular aminoacyl transfer steps of NRPS with aminolysis rate enhancements in neutral aqueous solutions of up to 5400-fold (k(cat)/k(uncat)). The catalysts operate as noncovalently associated peptide assemblies with composite active sites fashioned at the interface between helical subunits. Following the substrate loading at the active site cysteine, the juxtaposition of the resulting aminoacyl thiolester and the nucleophilic amine of the acyl acceptor moiety gives rise to high effective concentrations (up to 54 M) that facilitate interhelical aminoacyl transfer with rates typically exceeding 10(-4) sec(-1). Moreover, studies based on homo- and heteromeric assemblies, active site amino acid substitutions, kinetic analysis, and reaction modeling indicate that the de novo designed supramolecular catalysts reported herein exhibit some of the basic characteristics of natural enzymes, including precise positioning and pK(a) modulation of active site residues, covalent catalysis, and multiple product turnovers.