The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase.
The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase.
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DOI：
10.1021/acs.biochem.5b00945
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发表时间：
2016-02-23
期刊：
影响因子：
2.9
通讯作者：
Kohen A
中科院分区：
文献类型：
--
作者：
Francis K;Sapienza PJ;Lee AL;Kohen A
来自大肠杆菌的二氢叶酸还原酶（DHFR）长期以来一直作为模型酶来阐明蛋白质动力学和催化反应之间可能的联系。到目前为止，人类对应物的这些物理特性尚未得到严格的研究，但最近基于计算机的模拟表明，这两种DHFR在蛋白质动力学和催化的C-H→C氢化物转移步骤的紧密耦合程度上存在显着差异。为了测试这一预测，两个当代探针研究蛋白质动力学对催化的影响在这里结合：温度依赖性的内在动力学同位素效应（KIE）是敏感的物理性质的化学步骤，和蛋白质质量调制，减缓快速动态（飞秒至皮秒时间尺度）整个蛋白质。与E. coliDHFR，显示在5-45 °C范围内是温度无关的，表明在反应的过渡态（或隧穿就绪态- TRS）处供体和受体距离（DAD）的快速采样。这些酶的质量调制，通过同位素标记与13 C，15 N，和2 H在不可交换的氢产生重11%的酶。额外的质量对人酶的固有KIE没有影响。这一发现表明，人DHFR的质量调制既不影响DAD分布，也不影响DAD的构象采样动力学。此外，酶的周转数和产物的解离速率常数的减少表明，同位素取代影响的动力学步骤，而不是催化的C-H→C氢化物转移。研究结果进行了讨论，在快速动力学和它们在催化作用，计算和实验的比较，和同位素调制的重酶一般的解释。
Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H→C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs) that are sensitive to the physical nature of the chemical step, and protein mass-modulation that slows down fast dynamics (femto- to picosecond timescale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5–45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction’s transition state (or tunneling ready state – TRS). Mass modulation of these enzymes through isotopic labeling with 13C, 15N, and 2H at nonexchangeable hydrogens yield an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass-modulation of the human DHFR affects neither DAD distribution nor the DAD’s conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H→C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically-modulated heavy enzymes in general.