Muraki M, Harata K, Sugita N, Sato K
Biomolecules Department, National Institute of Bioscience and Human Technology, Ibaraki, Japan.
Biochemistry. 1996 Oct 22;35(42):13562-7. doi: 10.1021/bi9613180.
In order to reveal the origin of carbohydrate recognition specificity of human lysozyme by clarifying the difference in the binding mode of ligands in the active site, the inactivation of human lysozyme by 2',3'-epoxypropyl beta-glycoside derivatives of the disaccharides, N,N'-diacetylchitobiose [GlcNAc-beta-(1-->4)-GlcNAc] and N-acetyllactosamine [Gal-beta-(1-->4)-GlcNAc], was investigated and the three-dimensional structures of the affinity-labeled enzymes were determined by X-ray crystallography at 1.7 A resolution. Under the conditions comprising 2.0 x 10(-3) M labeling reagent and 1.0 x 10(-5) M human lysozyme at pH 5.4, 37 degrees C, the reaction time required to reduce the lytic activity against Micrococcus luteus cells to 50% of its initial activity was lengthened by 3.7 times through the substitution of the nonreducing end sugar residue, GlcNAc to Gal. The refined structure of human lysozyme labeled by 2',3'-epoxypropyl beta-glycoside derivatives of N,N'-diacetylchitobiose (HL/NAG-NAG-EPO complex) indicated that the interaction mode of the N,N'-diacetylchitobiose moiety in substites B and C in this study was essentially the same as in the case of the complex of human lysozyme with the free ligand. On the other hand, the hydrogen-bonding pattern and the stacking interaction at subsite B were remarkably different between the HL/NAG-NAG-EPO complex and human lysozyme labeled by the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine (HL/GAL-NAG-EPO complex). The reduced number of possible hydrogen bonds as well as the less favorable stacking between the side chain of Tyr63 in human lysozyme and the galactose residue in the HL/GAL-NAG-EPO complex reasonably explained the less efficient ability of the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine as compared to that of N,N'-diacetylchitobiose as an affinity labeling reagent toward human lysozyme.
为了通过阐明活性位点中配体结合模式的差异来揭示人溶菌酶碳水化合物识别特异性的起源，研究了二糖N，N'-二乙酰壳二糖[GlcNAc-β-(1→4)-GlcNAc]和N-乙酰乳糖胺[Gal-β-(1→4)-GlcNAc]的2'，3'-环氧丙基β-糖苷衍生物对人溶菌酶的失活作用，并通过X射线晶体学在1.7 Å分辨率下测定了亲和标记酶的三维结构。在pH 5.4、37℃条件下，标记试剂浓度为2.0×10⁻³ M、人溶菌酶浓度为1.0×10⁻⁵ M时，通过将非还原端糖残基从GlcNAc替换为Gal，使针对藤黄微球菌细胞的溶菌活性降低至其初始活性50%所需的反应时间延长了3.7倍。N，N'-二乙酰壳二糖的2'，3'-环氧丙基β-糖苷衍生物标记的人溶菌酶（HL/NAG-NAG-EPO复合物）的精细结构表明，本研究中取代位点B和C中的N，N'-二乙酰壳二糖部分的相互作用模式与游离配体与人溶菌酶复合物的情况基本相同。另一方面，HL/NAG-NAG-EPO复合物与人溶菌酶被N-乙酰乳糖胺的2'，3'-环氧丙基β-糖苷标记（HL/GAL-NAG-EPO复合物）相比，位点B处的氢键模式和堆积相互作用存在显著差异。人溶菌酶中Tyr63侧链与HL/GAL-NAG-EPO复合物中半乳糖残基之间可能的氢键数量减少以及堆积不太有利，合理地解释了N-乙酰乳糖胺的2'，3'-环氧丙基β-糖苷与人溶菌酶相比，作为亲和标记试剂的效率较低。
