Regulation of hydrolysis and transglycosylation in lysozyme-catalytic reaction.

溶菌酶催化反应中水解和转糖基化的调节。

• 批准号: 05660111
• 负责人: TORIKATA Takao
• 金额: $ 1.34万
• 依托单位: Kyushu Tokai University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05660111/
• 关键词: Lysozyme; Chemical modification; NMR spectrum; Catalytic reaction; Lysozyme; reaction mechanism; subsite; binding

The mechanism for the high efficiency of transglycosylation of lysozyme seems not to have been fully explained. We attempted to clarify the reaction mechanism of lysozyme-catalyzed reaction as follows ; 1.Enzyme Activity of Avian Egg-White Lysozyme : In turkey, peafowl, and quail lysozymes, Asp101-Met105 in the loop region of hen lysozyme (HEL) were replaced by other amino acids. The reaction time-courses of each lysozyme with (GlcNAc) _5 were exhibited different profiles from those of HEL,lowering the substrate affinity at subsite A and/or B.The time-courses of guinea-hen lysozyme (GHL), which replaced Arg114 in subsite F of HEL by His, was the same profile as those of HEL,suggested that His114 in GHL participated in binding of sugar residue at subsite F in the same manner as Arg114 in HEL.2.Enzyme Activity of His114-modified GHL : His114 in GHL was modified by DEP and monoiodoacetic acid. The projiles of time-courses of modified GHL deffered greatly from those of native lysozyme. The time-courses of modified GHL indicated that modified GHL may have increased rate of hydrolysis in comparison with that of native lysozyme. 3.Binding Mode of (GlcNAc) _6 for Trp108 Ester GHL : The resonance of C2H of His114 in ^1H-NMR spectrum is suitable as a probe for investigation of substrate binding at subsite E and F.The modified lysozyme in which an ester linkage had been formed between Glu35 and Trp108 (Trp108 ester GHL) showed no significant activity, and unaffected the environment around subsites. The interaction of (GlcNAc) _6 with modified lysozyme was studied by ^1H-NMR spectra. The resonance of C2H of His114 in Trp108 ester GHL did not change upon the addition of (GlcNAc) _6. The results indicate that the substrate binds to left side of subsite E and F.On the basis of those results, the high efficiency of transglycosylation of lysozyme can be presumed by the formation of a 2 : 1 substrate-acceptor-enzyme complex.

溶菌酶高效率转糖基化的机制似乎还没有完全解释。我们试图阐明溶菌酶催化反应的机理如下：1.禽蛋-白溶菌酶的活性：在火鸡、孔雀和鹌鹑溶菌酶中，鸡溶菌酶(HEL)环区的Asp101-Met105被其他氨基酸取代。每种溶菌酶与(GlcNAc)_5的反应时间曲线与HEL的不同，降低了A和/或B亚位的底物亲和力。His取代HEL F亚位的Arg114的豚鼠溶菌酶(GHL)的反应时间曲线与HEL的相同，表明GHL中的His114以与HEL中的Arg114相同的方式参与F亚位糖残基的结合。经修饰的GHL与天然溶菌酶的时间历程有很大不同。改性GHL的时间进程表明，与天然溶菌酶相比，改性GHL可能具有更高的水解率。3.Trp108 Ester GHL与(GlcNAc)_6的结合方式：His114的~1H-CNMR谱中C2H的共振峰适合作为研究E和F亚基结合底物的探针。修饰后的溶菌酶在Glu35和Trp108(Trp108 Ester GHL)之间形成了酯键，没有明显的活性，也不影响亚基周围的环境。用1H-核磁共振波谱研究了(GlcNAc)6与修饰溶菌酶的相互作用。His114在Trp108酯GHL中的C2H共振不随(GlcNAc)_6的加入而改变。结果表明，底物与E亚基和F亚基的左侧结合。在这些结果的基础上，可以推测溶菌酶的糖基化反应是通过形成2：1的底物-受体-酶复合体来实现的。