Regulation of hydrolysis and transglycosylation in lysozyme-catalytic reaction.

溶菌酶高效转糖基化的机制似乎还没有得到充分的解释。我们试图阐明溶菌酶催化反应的反应机理如下：1.禽蛋清溶菌酶的酶活性：在火鸡、孔雀和鹌鹑溶菌酶中，母鸡溶菌酶（HEL）环区的Asp101-Met105被其他氨基酸取代。与（GlcNAc） _5结合的溶菌酶的反应时间过程与HEL的不同，降低了A和/或b亚位点的底物亲和力，而用His取代HEL F亚位点Arg114的豚鼠-母鸡溶菌酶（GHL）的反应时间过程与HEL相同，说明GHL中的His114与HEL中的Arg114一样参与了F亚位点糖残基的结合。His114修饰GHL的酶活性：用DEP和单碘乙酸修饰GHL中的His114。与天然溶菌酶相比，改性GHL酶的时程分布有较大差异。改性GHL的时间过程表明，与天然溶菌酶相比，改性GHL的水解速率可能有所提高。3.（GlcNAc） _6与Trp108酯GHL的结合模式：His114的C2H共振在^1H-NMR谱中适合作为研究底物在E和f亚位点结合的探针。在Glu35和Trp108之间形成酯链的修饰溶菌酶（Trp108酯GHL）没有明显的活性，并且不影响亚位点周围的环境。用核磁共振谱研究了（GlcNAc） _6与改性溶菌酶的相互作用。（GlcNAc） _6的加入没有改变His114在Trp108酯GHL中的C2H共振。结果表明，底物与E和f亚位左侧结合。根据这些结果，可以通过形成2:1底物-受体-酶复合物来推测溶菌酶的高效转糖基化。

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.