Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in Environment, Faculty of Science, Masaryk University, Kamenice 5/A13, 62500 Brno, Czech Republic.
Biochem J. 2011 Apr 15;435(2):345-54. doi: 10.1042/BJ20101405.
An enzyme's substrate specificity is one of its most important characteristics. The quantitative comparison of broad-specificity enzymes requires the selection of a homogenous set of substrates for experimental testing, determination of substrate-specificity data and analysis using multivariate statistics. We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally useful substrates in the assessment of haloalkane dehalogenase activity are 1-bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using principal component analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinBDbjA>DhlADhaADbeADmbA>DatADmbCDrbA. After transforming the data, we were able to classify haloalkane dehalogenases into four SSGs (substrate-specificity groups). These functional groups are clearly distinct from the evolutionary subfamilies, suggesting that phylogenetic analysis cannot be used to predict the substrate specificity of individual haloalkane dehalogenases. Structural and functional comparisons of wild-type and mutant enzymes revealed that the architecture of the active site and the main access tunnel significantly influences the substrate specificity of these enzymes, but is not its only determinant. The identification of other structural determinants of the substrate specificity remains a challenge for further research on haloalkane dehalogenases.
酶的底物特异性是其最重要的特征之一。广谱酶的定量比较需要选择一组同质的底物进行实验测试，测定底物特异性数据，并使用多元统计进行分析。我们描述了对 9 种野生型和 4 种工程化卤代烷脱卤酶的底物特异性的系统分析。这些酶使用一套 30 种底物进行了实验表征，这些底物是使用统计实验设计从近 200 种卤代化合物中选择的。对活性数据的分析表明，在评估卤代烷脱卤酶活性时，最普遍有用的底物是 1-溴丁烷、1-碘丙烷、1-碘丁烷、1,2-二溴乙烷和 4-溴丁腈。使用主成分分析探索了酶之间的功能关系。对未转换的比活性数据的分析表明，野生型卤代烷脱卤酶的总体活性按以下顺序降低：LinBDbjA>DhlADhaADbeADmbA>DatADmbCDrbA。在对数据进行转换后，我们能够将卤代烷脱卤酶分为四个 SSG（底物特异性组）。这些功能组与进化亚家族明显不同，表明系统发育分析不能用于预测个别卤代烷脱卤酶的底物特异性。野生型和突变酶的结构和功能比较表明，活性位点和主入口隧道的结构显著影响这些酶的底物特异性，但不是其唯一决定因素。其他结构决定因素的确定仍然是进一步研究卤代烷脱卤酶的挑战。
