Teshima K, Kuramitsu S, Hamaguchi K, Sakiyama F, Mizuno K, Yamasaki N
J Biochem. 1980 Apr;87(4):1015-27.
The interactions of beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which Trp 62 is modified to kynurenine (Kyn 62-lysozyme) were studied by measuring the changes in the CD band and fluorescence due to the kynurenine at various pH values. The pH profiles of the binding constants of these saccharides to Kyn 62-lysozyme were very similar to those to intact lysozyme, although the binding constants were lower for the modified lysozyme than for intact lysozyme. The pK values of Asp 52, Glu 35, Asp 48, and Asp 101 in Kyn 62-lysozyme and in its complexes with beta-methyl-GlcNAc and with (GlcNAc)2 were in agreement with those of intact lysozyme and its complexes. The pK values of Asp 52 and Glu 35 in the modified lysozyme-(GlcNAc)3 complex were also in agreement with those of the complex with intact lysozyme, but the pK shift of Asp 101 was smaller for Kyn 62-lysozyme than for intact lysozyme. The significance of the decreased binding constants to Kyn 62-lysozyme is discussed. The pH dependence of the CD band due to the kynureinine in Kyn 62-lysozyme was interpreted in terms of the participation of the catalytic groups, Asp 52 (apparent pK 3.5) and Glu 35 (apparent pK 6.0), and the amino group of the kynurenine (apparent pK 0.75). This indicates that the ionization of the catalytic groups affects the state around Trp 62 and supports the previous proposal that there is a relation between the state around Trp 62 and the ionization of Glu 35 (Ikeda, K. & Hamaguchi, K. (1973) J. Biochem. 74, 221--230; (1975) ibid. 77, 1--16; Nakae et al. (1975) J. Biochem. 77, 993--1006). The intrinsic pK value of the amino group of the kynurenine at position 62 shifted from 2.1 to 0.8 on complexing with (GlcNAc)3, indicating between the kynurenine and the sugar residue at subsite B.
通过测量在不同pH值下由于犬尿氨酸导致的圆二色光谱带（CD band）和荧光的变化，研究了β-甲基-GlcNAc、(GlcNAc)2和(GlcNAc)3与鸡卵清溶菌酶[EC 3.2.1.17]（其中Trp 62被修饰为犬尿氨酸（Kyn 62-溶菌酶））之间的相互作用。这些糖类与Kyn 62-溶菌酶的结合常数的pH曲线与它们与完整溶菌酶的pH曲线非常相似，尽管修饰后的溶菌酶的结合常数低于完整溶菌酶。Kyn 62-溶菌酶及其与β-甲基-GlcNAc和(GlcNAc)2形成的复合物中Asp 52、Glu 35、Asp 48和Asp 101的pK值与完整溶菌酶及其复合物的pK值一致。修饰后的溶菌酶-(GlcNAc)3复合物中Asp 52和Glu 35的pK值也与完整溶菌酶复合物的pK值一致，但Kyn 62-溶菌酶中Asp 101的pK位移比完整溶菌酶中的小。讨论了与Kyn 62-溶菌酶结合常数降低的意义。Kyn 62-溶菌酶中由于犬尿氨酸导致的CD光谱带的pH依赖性是根据催化基团Asp 52（表观pK 3.5）和Glu 35（表观pK 6.0）以及犬尿氨酸的氨基（表观pK 0.75）的参与来解释的。这表明催化基团的电离影响Trp 62周围的状态，并支持先前提出的Trp 62周围的状态与Glu 35的电离之间存在关系的提议（池田，K.和滨口，K.（1973年）《生物化学杂志》74，221 - 230；（1975年）同上，77，1 - 16；中江等人（1975年）《生物化学杂志》77，993 - 1006）。与(GlcNAc)3络合时，62位犬尿氨酸氨基的固有pK值从2.1变为0.8，表明犬尿氨酸与亚位点B处的糖残基之间存在相互作用。
