Hadfield A T, Harvey D J, Archer D B, MacKenzie D A, Jeenes D J, Radford S E, Lowe G, Dobson C M, Johnson L N
Oxford Centre for Molecular Sciences, University of Oxford, U.K.
J Mol Biol. 1994 Nov 11;243(5):856-72. doi: 10.1006/jmbi.1994.1688.
The crystal structure of a mutant hen egg white lysozyme, in which the key catalytic residue aspartic acid 52 has been changed to a serine residue (D52S HEWL), has been determined and refined to a crystallographic R value of 0.173 for all data F > 0 between 8 and 1.9 A resolution. The D52S HEWL structure is very similar to the native HEWL structure (r.m.s. deviation of main-chain atoms 0.20 A). Small shifts that result from the change in hydrogen bonding pattern on substitution of Asp by Ser were observed in the loop between beta-strands in the region of residues 46 to 49. D52S HEWL exhibits less than 1% activity against the bacterial cell wall substrate. Cocrystallisation experiments with the hexasaccharide substrate beta(1-4) polymer of N-acetyl-D-glucosamine (GlcNAc6) resulted in crystals between 5 days and 14 days after the initial mixing of enzyme and substrate. Analysis by laser absorption mass spectrometry of the oligosaccharides present after incubation with native and D52S HEWL under conditions similar to those used for crystal growth showed that after 14 days with native HEWL complete catalysis to GlcNAc3. GlcNAc2 and GlcNac had occurred but with D52S HEWL only partial catalysis to the major products GlcNAc4 and GlcNAc2 had occurred and at least 50% of the GlcNAc6 remained intact. X-ray analysis of the D52S-oligosaccharide complex crystals showed that they contained the product GlcNAc4. The structure of the D52S HEWL-GlcNAc4 complex has been determined and refined to an R value of 0.160 for data between 8 and 2 A resolution. GlcNAc4 occupies sites A to D in the active site cleft. Careful refinement and examination of 2Fo-Fc electron density maps showed that the sugar in site D has the sofa conformation, a conformation previously observed with the HEWL complex with tetra-N-acetylglucosamine lactone transition state analogue, the HEWL complex with the cell wall trisaccharide and the phage T4 lysozyme complex with a cell wall product. The semi-axial C(5)-C(6) geometry of the sofa is stabilised by hydrogen bonds from the O-6 hydroxyl group to the main-chain N of Val109 and main-chain O of Ala107. The sugar in site D adopts the alpha configuration, seemingly in conflict with the observation that the hydrolysis of beta (1-4) glycosidie linkage by HEWL proceeds with 99.9% retention of beta-configuration.(ABSTRACT TRUNCATED AT 400 WORDS)
已确定一种突变型鸡卵清溶菌酶（其中关键催化残基天冬氨酸52已被替换为丝氨酸残基，即D52S HEWL）的晶体结构，并将其精修至所有8至1.9 Å分辨率下F > 0数据的晶体学R值为0.173。D52S HEWL结构与天然HEWL结构非常相似（主链原子的均方根偏差为0.20 Å）。在46至49位残基区域的β链之间的环中观察到由于丝氨酸取代天冬氨酸导致氢键模式变化而产生的小位移。D52S HEWL对细菌细胞壁底物的活性小于1%。用六糖底物N-乙酰-D-葡萄糖胺（GlcNAc6）的β(1-4)聚合物进行共结晶实验，在酶和底物初始混合后5天至14天内得到晶体。在与晶体生长所用条件相似的条件下，用天然HEWL和D52S HEWL孵育后，通过激光吸收质谱分析存在的寡糖，结果表明用天然HEWL孵育14天后已完全催化生成GlcNAc3、GlcNAc2和GlcNAc，但用D'52S HEWL仅部分催化生成主要产物GlcNAc4和GlcNAc2，且至少50%的GlcNAc6保持完整。D52S - 寡糖复合物晶体的X射线分析表明它们含有产物GlcNAc4。已确定D52S HEWL - GlcNAc4复合物的结构，并将其精修至8至2 Å分辨率下数据的R值为0.160。GlcNAc4占据活性位点裂隙中的A至D位点。对2Fo - Fc电子密度图进行仔细精修和检查表明，位点D中的糖具有沙发构象，这种构象先前在与四 - N - 乙酰葡糖胺内酯过渡态类似物的HEWL复合物、与细胞壁三糖的HEWL复合物以及与细胞壁产物的噬菌体T4溶菌酶复合物中观察到。沙发构象的半轴向C(5) - C(6)几何结构通过从O - 6羟基到Val109的主链N和Ala107的主链O的氢键得以稳定。位点D中的糖采用α构型，这似乎与观察到的HEWL对β(1 - 4)糖苷键的水解以99.9%的β构型保留率进行相矛盾。（摘要截断于400字）
