Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: comparison of wild-type protein and active-site mutant D27E.
Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: comparison of wild-type protein and active-site mutant D27E.
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DOI：
10.1016/j.bbapap.2013.09.024
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发表时间：
2013-12
期刊：
影响因子：
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通讯作者：
E. Ohmae;Y. Miyashita;S. Tate;K. Gekko;Soichiro Kitazawa;R. Kitahara;K. Kuwajima
中科院分区：
文献类型：
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作者：
E. Ohmae;Y. Miyashita;S. Tate;K. Gekko;Soichiro Kitazawa;R. Kitahara;K. Kuwajima
为了研究溶剂环境对大肠杆菌氢叶酸还原酶（DHFR）酶促功能的影响，我们比较了野生型蛋白与活性位点突变体D27E的酶促功能的盐依赖性、pH依赖性和压力依赖性，以及它们的结构和稳定性。酶活性的盐浓度依赖性表明，在中性ph下，无机阳离子与野生型DHFR结合并抑制其活性。野生型DHFR -叶酸二元配合物的1h - 15n HSQC光谱显示，阳离子结合位点位于Met20环附近。D27E突变体对一价阳离子不敏感，其酶活性的最佳pH值降低，底物二氢叶酸的kmandkdvalue增加，这表明该突变体的底物结合间隙略微打开，使活性位点侧链暴露在溶剂中。突变体展开引起的荧光强度的轻微增加和体积变化的减少也支持这种结构变化或修饰的空腔和水合作用。令人惊讶的是，当压力达到250 MPa时，突变体的酶活性增加，负激活体积为- 4.0或- 4.8 mL/mol（取决于溶剂系统），而野生型的酶活性降低，正激活体积为6.1或7.7 mL/mol。这些结果清楚地表明，在活性位点插入单个亚甲基可以显著改变DHFR的酶促反应机制，溶剂环境对该酶的功能起着重要作用。
To investigate the contribution of solvent environments to the enzymatic function ofEscherichia colidihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl2concentration-dependence of the1H–15N HSQC spectra of the wild-type DHFR–folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increasedKmandKdvalues for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250 MPa together with negative activation volumes of − 4.0 or − 4.8 mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7 mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme.