Quantitative analysis of substrate specificity of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26
Quantitative analysis of substrate specificity of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26
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DOI：
10.1021/bi047912o
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发表时间：
2005-03-08
期刊：
影响因子：
2.9
通讯作者：
Damborsky, J
中科院分区：
文献类型：
--
作者：
Kmunícek, J;Hynková, K;Damborsky, J
卤代烷烃脱卤酶是在卤代化合物中切割碳卤键的微生物酶。从少动鞘单胞菌UT26中分离得到的卤烷脱卤酶LinB是一种广谱特异性酶。用LinB酶对55种卤代脂肪烃和环烃进行了脱卤实验。使用统计实验设计系统地选择用于测试的化合物。测定了25个底物的稳态动力学常数k -m和k(cat)，这些底物可以被酶和低非生物水解检测到。经典的定量构效关系（qsar）用于将动力学常数与分子描述符联系起来，并产生了一个模型，该模型解释了94%的实验数据变异性。测试底物对这种卤烷脱卤酶的结合亲和力与疏水性、分子表面、偶极矩和体积：表面比相关。底物分子在LinB活性位点口袋中的结合与分子的大小呈非线性关系。结合亲和力随着底物大小的增加而增加，直到6个碳原子的链长，然后降低。然后使用比较结合能（COMBINE）分析来鉴定LinB中调节其底物特异性的氨基酸残基。一个具有三个统计显著主成分的模型解释了95%的实验数据变异性。底物分子与酶之间的范德华相互作用主导了COMBINE模型，这与底物大小在经典QSAR模型中的重要性一致。只有有限数量的蛋白质残基（6-8%）对解释结合亲和力的可变性有重要作用。对解释结合亲和性变异性重要的氨基酸残基如下：(i)第一壳残基Asn38、Asp108、Trp109、Glu132、Ile134、Phe143、Phe151、Phe169、Val173、Trp、207、Pro208、Ile211、Leu248和His272， （ii）通道残基Pro144、Asp147、Leu177和Ala247，以及（iii）第二壳残基Pro39和Phe273。隧道和第二壳残基代表了调节特异性的最佳靶点，因为它们的替换不会通过破坏活性位点结构而导致功能丧失。在定量比较两种蛋白家族成员模型的基础上，探讨了不同特异性的分子适应机制。
Haloalkane dehalogenases are microbial enzymes that cleave a carbon-halogen bond in halogenated compounds. The haloalkane dehalogenase LinB, isolated from Sphingomonas paucimobilis UT26, is a broad-specificity enzyme. Fifty-five halogenated aliphatic and cyclic hydrocarbons were tested for dehalogenation with the LinB enzyme. The compounds for testing were systematically selected using a statistical experimental design. Steady-state kinetic constants K-m and k(cat) were determined for 25 substrates that showed detectable cleavage by the enzyme and low abiotic hydrolysis. Classical quantitative structureactivity relationships (QSARs) were used to correlate the kinetic constants with molecular descriptors and resulted in a model that explained 94% of the experimental data variability. The binding affinity of the tested substrates for this haloalkane dehalogenase correlated with hydrophobicity, molecular surface, dipole moment, and volume:surface ratio. Binding of the substrate molecules in the active site pocket of LinB depends nonlinearly on the size of the molecules. Binding affinity increases with increasing substrate size up to a chain length of six carbon atoms and then decreases. Comparative binding energy (COMBINE) analysis was then used to identify amino acid residues in LinB that modulate its substrate specificity.A model with three statistically significant principal components explained 95% of the experimental data variability. van der Waals interactions between substrate molecules and the enzyme dominated the COMBINE model, in agreement with the importance of substrate size in the classical QSAR model. Only a limited number of protein residues (6-8%) contribute significantly to the explanation of variability in binding affinities. The amino acid residues important for explaining variability in binding affinities are as follows: (i) first-shell residues Asn38, Asp108, Trp109, Glu132, Ile134, Phe143, Phe151, Phe169, Val173, Trp,207, Pro208, Ile211, Leu248, and His272, (ii) tunnel residues Pro144, Asp147, Leu177, and Ala247, and (iii) second-shell residues Pro39 and Phe273. The tunnel and the second-shell residues represent the best targets for modulating specificity since their replacement does not lead to loss of functionality by disruption of the active site architecture. The mechanism of molecular adaptation toward a different specificity is discussed on the basis of quantitative comparison of models derived for two protein family members.