Protonation states of hen egg-white lysozyme observed using D/H contrast neutron crystallography
Protonation states of hen egg-white lysozyme observed using D/H contrast neutron crystallography
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D/H 对比中子晶体学观察鸡蛋清溶菌酶的质子化状态
DOI：
10.1107/s2059798322004521
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Fujiwara Satoru
中科院分区：
文献类型：
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作者：
Chatake Toshiyuki;Tanaka Ichiro;Kusaka Katsuhiro;Fujiwara Satoru
鸡蛋清溶菌酶（HEWL）是一种酶蛋白，其活性部位含有两个酸性氨基酸Glu 35和Asp52。Glu35作为质子供体的基板和Asp52与带正电的基板相互作用，这表明这些残基的不同质子化状态。然而，迄今为止，中子晶体学的研究还没有提供一个一致的图片这些残留物的质子化状态。只有一项研究成功地观察到Glu35和Asp52在三斜晶系中的活性质子化状态。然而，它们在最广泛研究的四元晶系中的活性状态仍然未知。D/H对比技术在中子晶体学中的应用提高了定位蛋白质中可交换D/H原子的能力。在本研究中，制备了D2O和H2O溶剂晶体。每个中子数据集收集只有五天，结合飞行时间衍射仪（iBIX）和日本质子加速器研究综合体的spiral中子源。D/H对比图提供了更好的可视化的D/H原子在HEWL比传统的中子散射长度密度图。中子D/H对比图表明，交替质子化的OE1和OE2原子的羧基的Glu 35。这种替代质子化在不存在底物的情况下发生，其中不发生质子化位点的高选择性。在这种情况下，只有OE1-HE1键在OE1-HE1和OE2-HE2之间的平衡中攻击底物，或者OE2-HE2键的H+离子在底物结合之前或之后移动到OE1原子以引发催化反应。相反，Asp52的羧基没有质子化。未观察到其他Asp和Glu残基的羧基质子化。这些结果与NMR光谱的结果一致，并解释了在脱辅基形式的HEWL的活性位点处的质子化状态。
Hen egg-white lysozyme (HEWL) is an enzymatic protein with two acidic amino acids, Glu35 and Asp52, in its active site. Glu35 acts as a proton donor to the substrate and Asp52 interacts with the positively charged substrate, suggesting different protonation states of these residues. However, neutron crystallographic studies thus far have not provided a consistent picture of the protonation states of these residues. Only one study succeeded in observing the active protonation states of Glu35 and Asp52 in the triclinic crystal system. However, their active states in the most widely studied tetragonal crystal system are still unknown. The application of the D/H contrast technique in neutron crystallography improves the ability to locate exchangeable D/H atoms in proteins. In the present study, D2O and H2O solvent crystals were prepared. Each neutron data set was collected for only five days by combining a time-of-flight diffractometer (iBIX) and the spallation neutron source at the Japan Proton Accelerator Research Complex. The D/H contrast map provided better visualization of the D/H atoms in HEWL than the conventional neutron scattering length density map. The neutron D/H contrast map demonstrated the alternative protonation of the OE1 and OE2 atoms in the carboxyl group of Glu35. This alternative protonation occurs in the absence of a substrate, where high selectivity of the protonation site does not occur. In this case, only the OE1—HE1 bond attacks the substrate in an equilibrium between OE1—HE1 and OE2—HE2, or the H+ ion of the OE2—HE2 bond moves to the OE1 atom just before or after substrate binding to initiate the catalytic reaction. In contrast, the carboxyl group of Asp52 is not protonated. Protonation of the carboxyl group was not observed for other Asp and Glu residues. These results are consistent with results from NMR spectroscopy and explain the protonation states at the active site in the apo form of HEWL.