DISSECTION OF PROTEIN-CARBOHYDRATE INTERACTIONS IN MUTANT HEN EGG-WHITE LYSOZYME COMPLEXES AND THEIR HYDROLYTIC ACTIVITY
DISSECTION OF PROTEIN-CARBOHYDRATE INTERACTIONS IN MUTANT HEN EGG-WHITE LYSOZYME COMPLEXES AND THEIR HYDROLYTIC ACTIVITY
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DOI：
10.1006/jmbi.1994.0139
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发表时间：
1995-03-24
影响因子：
5.6
通讯作者：
KUMAGAI, I
中科院分区：
文献类型：
--
作者：
MAENAKA, K;MATSUSHIMA, M;KUMAGAI, I
鸡蛋清溶菌酶的结合亚位点 B 中的 Trp62 显示出在蛋白质-碳水化合物相互作用中经常观察到的一般特征，包括与水分子的堆积相互作用和氢键网络。我们小组之前的一份报告表明，通过用酪氨酸或苯丙氨酸取代 Trp62 来扰动这些相互作用会影响底物结合模式，并增强水解活性。为了阐明这些蛋白质-碳水化合物相互作用的结构和功能变化之间的关系，在 1.8 埃分辨率下分析了与底物类似物 (GlcNAc)(3) 复合的 Trp62Tyr 和 Trp62Phe 突变体通过X射线晶体学。突变酶的总体结构与野生型酶的总体结构没有区别。尽管野生型酶仅以一种结合模式 (A-B-C) 结合 (GlcNAc)(3)，但 Trp62Tyr 突变体以两种结合模式 (A-B-C、B-C-D) 结合 (GlcNAc)(3)，并且 Trp62Phe 突变体具有更弱的结合模式。 Tyr62 和 Phe62 的芳香环保持与碳水化合物分子的相互作用，但与 B 位点中的 GlcNAc 的堆积相互作用比野生型酶少。 Tyr62 的羟基与水分子相互作用较弱，水分子介导 B 和 C 位点 GlcNAc 残基中的氢键。 C 位中 GlcNAc 残基的 C-6 羟基围绕 C-5-C-6 键旋转，以完成 Trp62Tyr 突变体-(GlcNAc)(3) 复合物中的氢键网络。另一方面，Trp62Phe 突变体-(GlcNAc)(3) 中不形成该氢键网络。除了这些结构研究之外，还确定了 4-甲基伞形基 N-乙酰基壳三糖 ((GlcNAc)(3)-MeU) 水解的动力学参数，以进一步表征这些突变型溶菌酶的酶性质。这表明氢键网络的调节，包括碳水化合物和水分子的柔性部分和/或 B 位点堆积相互作用的轻微减少，改变了与碳水化合物的结合模式并诱导水解活性的增强。
Trp62 in the binding subsite B of hen egg-white lysozyme shows general features often observed in protein-carbohydrate interactions including a stacking interaction and a hydrogen bonding network with water molecules. A previous report by our group showed that the perturbation of these interactions by substitution of Trp62 with tyrosine or phenylalanine affects the substrate binding modes and also enhances the hydrolytic activity In order to elucidate the relationship between structural and functional changes of these protein-carbohydrate interactions, the Trp62Tyr and Trp62Phe mutants complexed with the substrate analogue, (GlcNAc)(3), were analyzed at 1.8 Angstrom resolution by X-ray crystallography. The overall structures of the mutant enzymes are indistinguishable from that of the wild type enzyme. Although the wild-type enzyme binds (GlcNAc)(3) in only one binding mode (A-B-C), the Trp62Tyr mutant binds (GlcNAc)(3) in two binding modes (A-B-C, B-C-D) and the Trp62Phe mutant has an even weaker binding mode. The aromatic rings of Tyr62 and Phe62 maintain their interactions with the carbohydrate molecules, but make fewer stacking interactions with the GlcNAc in the B site than the wild-type enzyme does. The hydroxyl group of Tyr62 interacts weakly with a water molecule which mediates hydrogen bonding in the GlcNAc residues in the B and C sites. The C-6 hydroxyl group of the GlcNAc residue in the C site rotates around the C-5-C-6 bond to complete the hydrogen bond network in the Trp62Tyr mutant-(GlcNAc)(3) complex. On the other hand, this hydrogen bonding network does not form in the Trp62Phe mutant-(GlcNAc)(3). In addition to these structural studies, the kinetic parameters of the hydrolysis of 4-methylumbelliferyl N-acetyl-chitotriose, ((GlcNAc)(3)-MeU), have been determined in order to further characterize the enzymatic properties of these mutant lysozymes.This demonstrates that the modulation of the hydrogen bonding network, including the flexible part of the carbohydrate and water molecules and/or the slight reduction of stacking interaction in the B site, alters the binding mode toward the carbohydrate and induces an enhancement of the hydrolytic activity.