Regulation of hydrolysis and transglycosylation in lysozyme-catalytic reaction.

溶菌酶高效率转糖基化的机理似乎还没有完全解释。本研究试图阐明溶菌酶催化反应的反应机理如下：1.鸡蛋白溶菌酶的酶活性：在火鸡、孔雀和鹌鹑溶菌酶中，鸡溶菌酶（HEL）环区的Asp 101-Met 105被其它氨基酸取代。每种溶菌酶与（GlcNAc）_5的反应时程与HEL不同，降低了A和/或B亚位点的底物亲和力。用His取代HEL的F亚位点的Arg 114的豚鼠溶菌酶（GHL）的反应时程与HEL相同，表明GHL中的His 114以与HEL中的Arg 114相同的方式参与在亚位点F处的糖残基的结合。2. His 114修饰的GHL的酶活性：GHL中的His 114被DEP和一碘乙酸修饰。改性GHL与天然溶菌酶作用的时间进程有很大差异。改性GHL的时间进程表明，与天然溶菌酶相比，改性GHL可能具有增加的水解速率。3.（GlcNAc）_6与Trp 108酯GHL的结合方式：His 114的C_2H共振可作为研究底物在E和F亚位点结合的探针。Glu 35与Trp 108形成酯键的修饰溶菌酶（Trp 108酯GHL）活性不明显，且不影响亚位点周围的环境。用~ 1H-NMR研究了（GlcNAc）_6与修饰溶菌酶的相互作用。Trp 108酯GHL中His 114的C2 H的共振在加入（GlcNAc）_6后没有改变。结果表明，底物结合于E和F亚基的左侧，由此推测溶菌酶的转糖基化反应是通过形成2：1的底物-受体-酶复合物实现的。

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.