Kawamura Shunsuke, Toshima Gen, Imoto Taiji, Araki Tomohiro, Torikata Takao
Department of Bioscience, School of Agriculture, Kyushu Tokai University, Aso, Kumamoto 869-1404, Japan.
J Biochem. 2002 May;131(5):663-70. doi: 10.1093/oxfordjournals.jbchem.a003149.
We analyzed the enzymatic properties of duck egg-white lysozyme II (DEL), which differs from hen egg-white lysozyme (HEL) in nineteen amino acid substitutions. A substrate binding study showed that DEL binds to the substrate analog at subsites A-C in the same manner as HEL. However, the experimental time-courses of DEL against the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed remarkably enhanced production of (GlcNAc)(2) and reduced production of (GlcNAc)(1) as compared to in the case of HEL. Computer simulation of the DEL-catalyzed reaction suggested that the amino acid substitutions at subsites E and F (Phe34 to Tyr and Asn37 to Ser) caused the great alteration in the time-courses of DEL. Subsequently, the enzymatic reactions of mutants, in which Phe34 and Asn37 in HEL were converted to Tyr and Ser, respectively, were characterized. The time-courses of the F34Y mutant exhibited profiles similar to those of HEL. In contrast, the characteristics of the N37S mutant were different from those of HEL and rather similar to those of DEL; the order of the amounts of (GlcNAc)(1) and (GlcNAc)(2) was reversed in comparison with in the case of HEL. Enhanced production of (GlcNAc)(2) was also observed for the mutant protein, F34Y/N37S, with two substitutions. These results indicated that the substitution of Asn37 with Ser can account, at least in part, for the characteristic time-courses of DEL. Moreover, replacement of Asn37 with Ser reduced the rate constant of transglycosylation. The substitution of the Asn37 residue may affect the transglycosylation activity of HEL.
我们分析了鸭蛋清白溶菌酶II（DEL）的酶学特性，它与鸡蛋清白溶菌酶（HEL）在19个氨基酸替换上存在差异。一项底物结合研究表明，DEL与底物类似物在A - C亚位点的结合方式与HEL相同。然而，DEL作用于底物N - 乙酰葡糖胺五聚体（GlcNAc）5的实验时间进程显示，与HEL相比，（GlcNAc）2的生成显著增加，而（GlcNAc）1的生成减少。DEL催化反应的计算机模拟表明，E和F亚位点的氨基酸替换（Phe34突变为Tyr以及Asn37突变为Ser）导致了DEL时间进程的巨大改变。随后，对HEL中Phe34和Asn37分别被转换为Tyr和Ser的突变体的酶促反应进行了表征。F34Y突变体的时间进程表现出与HEL相似的特征。相比之下，N37S突变体的特征与HEL不同，且与DEL相当相似；与HEL相比，（GlcNAc）1和（GlcNAc）2的生成量顺序发生了逆转。对于具有两个替换位点的突变蛋白F34Y/N37S，也观察到了（GlcNAc）2生成的增加。这些结果表明，Asn37被Ser替换至少部分地解释了DEL的特征时间进程。此外，Asn37被Ser替换降低了转糖基化的速率常数。Asn37残基的替换可能会影响HEL的转糖基化活性。
