脱卤过氧化物酶(DHP)具有不同的催化活性,取决于底物结合构象,pH值,和血红素上方远端口袋的动力学。根据我们的假设,底物的分子结构和结合方向在DHP指导酶的功能。酶动力学研究表明,尽管序列同源性为96%,但DHPB的催化活性显着高于DHPA。有30多个具有DHPB的衬底结合结构,每个都提供了对活性位点酶促结合性质的了解。相比之下,在与DHPA的复合物中,小分子的唯一X-射线晶体结构是酚。这项研究的重点是研究DHPA中的底物结合,以与DHPB结构进行比较。选择已知与晶体中的DHPB结合的15个基底,以测试将基底浸泡到DHPA中是否会产生类似的结构。这些底物中的五个产生了与底物结合的DHPA的X射线晶体结构,即,2,4-二氯苯酚(1.48µ,PDB:8EJN),2,4-二溴苯酚(1.52µ,PDB:8VSK),4-硝基苯酚(2.03µ,PDB:8VKC),4-硝基儿茶酚(1.40µ,PDB:8VKD),和4-溴-邻甲酚(1.64贝达,PDB:8VZR)。对于与DHPB结合的剩余底物,如甲酚,5-溴吲哚,苯并咪唑,4,4-联苯酚,4.4-亚乙基双酚,2,4-二甲氧基苯酚,和愈创木酚,DHPA中的电子密度图不足以确定底物的存在,更不用说他们的方向了。在我们手中,只有酚类,4-Br-邻甲酚,和4-硝基儿茶酚可以浸入结晶DHPA中。没有观察到较大的底物结合。选择最少7个悬滴用于浸泡,每种基质筛选超过50个晶体。直接比较同一底物在DHPA和B中的结合模式的五个高质量实例进一步支持了底物结合构象决定DHP酶功能的假设。
Dehalperoxidase (DHP) has diverse catalytic activities depending on the substrate binding conformation, pH, and dynamics in the distal pocket above the heme. According to our hypothesis, the molecular structure of the substrate and binding orientation in DHP guide enzymatic function. Enzyme kinetic studies have shown that the catalytic activity of DHP B is significantly higher than that of DHP A despite 96% sequence homology. There are more than 30 substrate-bound structures with DHP B, each providing insight into the nature of enzymatic binding at the active site. By contrast, the only X-ray crystallographic structures of small molecules in a complex with DHP A are phenols. This study is focused on investigating substrate binding in DHP A to compare with DHP B structures. Fifteen substrates were selected that were known to bind to DHP B in the crystal to test whether soaking substrates into DHP A would yield similar structures. Five of these substrates yielded X-ray crystal structures of substrate-bound DHP A, namely, 2,4-dichlorophenol (1.48 Å, PDB: 8EJN), 2,4-dibromophenol (1.52 Å, PDB: 8VSK), 4-nitrophenol (2.03 Å, PDB: 8VKC), 4-nitrocatechol (1.40 Å, PDB: 8VKD), and 4-bromo-o-cresol (1.64 Å, PDB: 8VZR). For the remaining substrates that bind to DHP B, such as cresols, 5-bromoindole, benzimidazole, 4,4-biphenol, 4.4-ethylidenebisphenol, 2,4-dimethoxyphenol, and guaiacol, the electron density maps in DHP A are not sufficient to determine the presence of the substrates, much less their orientation. In our hands, only phenols, 4-Br-o-cresol, and 4-nitrocatechol can be soaked into crystalline DHP A. None of the larger substrates were observed to bind. A minimum of seven hanging drops were selected for soaking with more than 50 crystals screened for each substrate. The five high-quality examples of direct comparison of modes of binding in DHP A and B for the same substrate provide further support for the hypothesis that the substrate-binding conformation determines the enzyme function of DHP.