Effects of five-tryptophan mutations on structure, stability and function of Escherichia coli dihydrofolate reductase
Effects of five-tryptophan mutations on structure, stability and function of Escherichia coli dihydrofolate reductase
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DOI：
10.1093/oxfordjournals.jbchem.a003004
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发表时间：
2001-09-01
影响因子：
2.7
通讯作者：
Gekko, K
中科院分区：
文献类型：
--
作者：
Ohmae, E;Sasaki, Y;Gekko, K
为了阐明色氨酸残基在大肠杆菌二氢叶酸还原酶（DHFR）结构、稳定性和功能中的作用，将DHFR的5个色氨酸残基分别用亮氨酸、苯丙氨酸或缬氨酸进行定点突变（W22 F、W22 L、W30 L、W 47 L、W74 F、W74 L、W133 F和W133 V）。这些突变体的远紫外圆二色性（CD）光谱显示，激子之间的耦合Trp 47和Trp 74强烈影响野生型DHFR的肽CD，和Trp 133也有明显的贡献。未观察到单个色氨酸残基对野生型DHFR的荧光光谱的贡献的加和性，Trp 74具有显性效应。这些单色氨酸突变诱导尿素解折叠自由能的大的变化，其值为1.79-7.14 kcal/mol，而野生型DHFR的值为6.08 kcal/mol。CD和荧光光谱的分析表明，热去折叠涉及一个中间体与天然样的二级结构，中断的Trp 47-Trp 74激子耦合，和溶剂暴露的Trp 30和Trp 47侧链。除W22 L（13%）外，所有突变体均保留野生型DHFR酶活性的50%以上。这些结果表明，DBFR的5个色氨酸残基在其结构和稳定性中起重要作用，但并不影响其酶功能。
To elucidate the roles of tryptophan residues in the structure, stability, and function of Escherichia coli dihydrofolate reductase (DHFR), its five tryptophan residues were replaced by site-directed mutagenesis with leucine, phenylalanine or valine (W22F, W22L, W30L, W47L, W74F, W74L, W133F, and W133V). Far-ultraviolet circular dichroism (CD) spectra of these mutants reveal that exciton coupling between Trp47 and Trp74 strongly affects the peptide CD of wild-type DHFR, and that Trp133 also contributes appreciably. No additivity was observed in the contributions of individual tryptophan residues to the fluorescence spectrum of wild-type DHFR, Trp74 having a dominant effect. These single-tryptophan mutations induce large changes in the free energy of urea unfolding, which showed values of 1.79-7.14 kcal/mol, compared with the value for wild-type DHFR of 6.08 kcal/mol. Analysis of CD and fluorescence spectra suggests that thermal unfolding involves an intermediate with the native-like secondary structure, the disrupted Trp47-Trp74 exciton coupling, and the solvent-exposed Trp30 and Trp47 side chains. All the mutants except W22L (13%) retain more than 50% of the enzyme activity of wild-type DHFR. These results demonstrate that the five tryptophan residues of DBFR play important roles in its structure and stability but do not crucially affect its enzymatic function.