Yang Y, Hamaguchi K
J Biochem. 1980 Apr;87(4):1003-14.
The binding constants of 4-methylumbelliferyl-beta-glycosides of (GlcNAc)2 ((GlcNAc)2-MeU) and of (GlcNAc)3 ((GlcNAc)3-MeU) to hen lysozyme [EC 3.2.1.17] were determined by measuring changes in the fluorescence at 375 nm. It was shown that (GlcNAc)2-MeU and (GlcNAc)3-MeU bind mainly at subsites B, C, and D, and A, B, C, and D, respectively, with the terminal MeU group bound at subsite D. The rate of hydrolysis of (GlcNAc)3-MeU catalyzed by hen and turkey lysozymes was determined at 0.1 ionic strength and 42 degrees C in the pH range of 2 to 8. The release of 4-methylumbelliferone was followed fluorimetrically. The pH dependences of kcat, kcat/Km, and Km were analyzed assuming that that nonproductive binding occurs competitively and that the molecular species with ionized Asp 52 and protonated Glu 35 is active. Comparison of the pH dependences of the kinetic constants for hen lysozyme with those for turkey lysozyme, in which Asp 101 of hen lysozyme is replaced by Gly, made it possible to determine the pK values of Asp 52, Glu 35, and Asp 101. The pK values of Asp 52 and Glu 35 were 3.60 and 6.20, respectively, for hen and turkey lysozymes and 3.95 and 6.55, respectively, for their nonproductive complexes. The pK value of Asp 101 of hen lysozyme was 4.20 for the free enzyme, 3.30 for the nonproductive complex, and 3.95 for the productive complex. These pK values, except for the pK value of Asp 52 of the nonproductive complex, are in excellent agreement with those determined by spectroscopic methods in our laboratory (Kurasmitsu et al. (1974) J. Biochem. 76, 671--683; (1975) ibid. 77, 291--301; (1977) ibid. 82, 585--597; (1978) ibid. 83, 159--170). This demonstrates that lysozyme-catalyzed hydrolysis can be fully explained in terms of the proposal based on the X-ray data (Blake et al. (1967) Proc. Roy. Soc. B167, 378--388), with regard to the participation of Asp 52 and Glu 35.
通过测量375nm处荧光的变化，测定了（GlcNAc）2（（GlcNAc）2-MeU）和（GlcNAc）3（（GlcNAc）3-MeU）的4-甲基伞形酮基-β-糖苷与鸡溶菌酶[EC 3.2.1.17]的结合常数。结果表明，（GlcNAc）2-MeU和（GlcNAc）3-MeU分别主要结合在亚位点B、C和D以及A、B、C和D，末端MeU基团结合在亚位点D。在0.1离子强度和42℃下，在pH 2至8范围内测定了鸡和火鸡溶菌酶催化（GlcNAc）3-MeU水解的速率。通过荧光法跟踪4-甲基伞形酮的释放。假设非生产性结合具有竞争性，并且离子化的Asp 52和质子化的Glu 35的分子物种具有活性，分析了kcat、kcat/Km和Km的pH依赖性。将鸡溶菌酶与火鸡溶菌酶（其中鸡溶菌酶的Asp 101被Gly取代）的动力学常数的pH依赖性进行比较，从而确定Asp 52、Glu 35和Asp 101的pK值。鸡和火鸡溶菌酶的Asp 52和Glu 35的pK值分别为3.60和6.20，其非生产性复合物的pK值分别为3.95和6.55。鸡溶菌酶的Asp 101的pK值在游离酶中为4.20，在非生产性复合物中为3.30，在生产性复合物中为3.95。除了非生产性复合物中Asp 52的pK值外，这些pK值与我们实验室通过光谱方法测定的值（Kurasmitsu等人（1974年）《生物化学杂志》76卷，671 - 683页；（1975年）同上，77卷，291 - 301页；（1977年）同上，82卷，585 - 597页；（1978年）同上，83卷，159 - 170页）非常一致。这表明，就Asp 52和Glu 35的参与而言，溶菌酶催化的水解可以根据基于X射线数据的提议（Blake等人（1967年）《皇家学会学报》B167卷，378 - 388页）得到充分解释。
