Binding site optimisation for artificial enzymes by diffusion NMR of small molecules
Binding site optimisation for artificial enzymes by diffusion NMR of small molecules
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DOI：
10.1002/chem.200390195
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发表时间：
2003-04-14
影响因子：
4.3
通讯作者：
Motherwell, WB
中科院分区：
文献类型：
--
作者：
Atkinson, CE;Aliev, AE;Motherwell, WB
提出了一种基于扩散核磁共振“小分子-小分子”结合研究的人工酶结合部位优化方法。由于所选择的反应是酯(1-([4-(4-carboxy-1-oxobutyl)-aminobenzyl]-phenethyl酯)的水解，所以选择了类似的磷酸酯(2-([4-(4-carboxy-1-oxobutyl)-aminobenzyl]-phosphonic-苯乙酯)作为合适的过渡态类似物。核磁共振研究的关键目标是找到一个具有能够与TSA的酸性中心结合的官能团的单元。使用了9个主要含有碱性和羟基的二肽，通过脉冲场梯度核磁共振测定结合时扩散系数D(PEP)的变化来研究它们与TSA的亲和力。测定了298K时D(PEP)在D(2O)中Pd 5，7和10的值，以及含有一个二肽和TSA的混合物中的D(PEP)。由于这两个组分都是M<；500的低分子物种，TSA与二肽的比例为10：1被用来检测D(PEP)的显著变化。结果表明，在水溶液中，含碱性残基的二肽对TSA的亲和力高于含羟基或脂肪族侧链的二肽。D(PEP)相对变化最显著的是H-Arg-Arg-OH，通过测定不同浓度TSA的D(PEP)，估计其结合常数为86Lm(-1)。此外，还研究了TSA与具有聚烯丙基胺骨架和随机分布的Arg-Arg结合位点的新型水溶性聚合物的结合，结合常数估计大于或等于1500Lm(-1)。进一步的催化活性测试证实，以Arg-Arg为结合位点的聚合物能够显著加快酯1的水解速度。
A binding site optimisation protocol for the design of artificial enzymes based on "small molecule-small molecule" binding studies by diffusion NMR is presented. Since the reaction chosen was the hydrolysis of ester 1 ([4-(4-carboxy-1-oxobutyl)-aminobenzyl]-phenethyl ester), an analogous phosphonate ester 2 ([4-(4-carboxy-1-oxobutyl)-aminobenzyl]-phosphonic phenethyl ester) was selected as a suitable transition state analogue (TSA). The key objective of the NMR studies was to find a unit with functional groups capable of binding to the acidic sites of the TSA. Nine dipeptides, mainly with basic and hydroxyl groups, were used and their affinity to the TSA was studied by measuring the change in the diffusion coefficient, D(pep), upon binding by pulse field gradient NMR. The value of D(pep) at 298 K in D(2)O at pD 5, 7 and 10 was measured both in free solution, and mixtures containing one dipeptide and the TSA. As both components are low molecular weight species with M < 500, a TSA-to-dipeptide ratio of 10:1 was used to detect significant changes in D(pep). The results revealed that dipeptides with basic residues show higher affinity to the TSA than those with hydroxyl or aliphatic side chains in aqueous solutions. The dipeptide showing the most significant relative change in D(pep) was H-Arg-Arg-OH, and the binding constant was estimated to be 86 LM(-1) by measuring D(pep) at varying concentrations of the TSA. In addition, binding of the TSA to a new water-soluble polymer with a polyallylamine backbone and randomly distributed Arg-Arg binding sites was examined, and the binding constant was estimated to be greater than or equal to 1500 LM(-1). As confirmed by further catalytic activity tests, polymers containing Arg-Arg as a binding site are capable of significant rate accelerations in the hydrolysis of ester 1.