Studies on lysozyme-catalyzed transglycosylation

溶菌酶是一种碳水解酶，具有很高的糖基化效率。为了阐明转糖基化的分子机制，以鸡溶菌酶为参照物，对几内亚溶菌酶进行了比较研究。鸡溶菌酶F亚位的&lt；Arg_(114)&gt；替换为豚鼠溶菌酶中的&lt；His_(114)&gt；。以壳五糖(GlcNAc)_5&GT为底物，测量了实验时间进程，并通过计算机分析估算了反应参数值。豚鼠-母鸡溶菌酶的时间进程特征是在反应早期增加~lt；(GlcNAc)_4&Gt；，这可能是由于转糖基化速率减慢所致。在不同的小灵通上测量实验的时间进程。推测转糖基化的pH依赖性是由于~lt；His_(114)&Gt；的电离状态所致。用二乙基焦碳酸酯(DEP)对豚鼠溶菌酶F亚基的~lt；His_(114)&gt；进行了化学修饰。实验结果表明，DEP-溶菌酶主要催化底物的水解，但几乎不催化转糖基化，由此推测受体分子和底物结合在F亚基的不同位置，推测溶菌酶-底物-受体复合体的形成导致了转糖基化的高效。

Lysozyme, which is a carbohydrolase, exhibits the high efficiency of transglycosylation. To elucidate the molecular mechanism of transglycosylation, guineahen lysozyme was subjected to a comparative study taking hen lysozyme as reference. <Arg_(114)> in subsite F of hen lysozyme is replaced with <His_(114)> in guinea-hen lysozyme. The experimental time-courses were measured with a substrate of chitopentaose <(GlcNAc)_5> and the values of reaction parameters were estimated from the experimental time-courses by computer analysis. The characteristic of the time-courses of guinea-hen lysozyme was increased the production of <(GlcNAc)_4> at an early stage of the reaction, which may be caused by the slowing down of the rate of transglycosylation. The experimental time-courses were measured at various pHs. It was assumed that pH-dependence of transglycosylation was due to the ionizable state of <His_(114)> . The chemical modification of <His_(114)> in subsite F of guinea-hen lysozyme was attempted with diethylpyrocarbonate (DEP). The experimental time-courses indicated that DEP-lysozyme catalyzed predominantly hydrolysis of substrate, but scarcely transglycosylation.From those results, acceptor molecule and substrate can be speculated to bind to a different position in subsite F. It is presumed that the formation of lysozyme-substrate-acceptor complex results in the high efficiency of transglycosylation.