The role of Arg114 at subsites E and F in reactions catalyzed by hen egg-white lysozyme
The role of Arg114 at subsites E and F in reactions catalyzed by hen egg-white lysozyme
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DOI：
10.1271/bbb.70694
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发表时间：
2008-03-01
影响因子：
1.6
通讯作者：
Torikata, Takao
中科院分区：
文献类型：
--
作者：
Kawamura, Shunsuke;Chijiiwa, Yuki;Torikata, Takao
为了更好地了解E和F亚位点在溶菌酶催化反应中的作用，我们制备了突变酶，其中位于蛋清溶菌酶（HEL）中E和F亚位点右侧的Arg114被Lys、His或Ala取代。用His或Ala替代Arg114会降低对人工底物乙二醇几丁质的水解活性，而用Lys替代则几乎没有影响。用底物n -乙酰氨基葡萄糖五聚体（GlcNAc）(5)进行的动力学分析表明，Arg残基的替换降低了E-F位点的结合自由能和转糖基化的速率常数。R114A的转糖基化速率常数约为野生型酶的一半。对R114H和R114A的H-1-NMR分析表明，突变引起的结构变化并不局限于Arg114周围区域，而是扩展到Phe34和Trp123的芳香侧链上，这些信号在野生型中通过核Overhauser效应（NOE）相互连接。我们推测，这种构象变化导致底物和受体在E和F亚位结合的差异，降低了溶菌酶糖基转移反应的效率。
To understand better the role of subsites E and F in lysozyme-catalyzed reactions, mutant enzymes, in which Arg114, located on the right side of subsites E and F in hen egg-white lysozyme (HEL), was replaced with Lys, His, or Ala, were prepared. Replacement of Arg114 with His or Ala decreased hydrolytic activity toward an artificial substrate, glycol chitin, while replacement with Lys had little effect. Kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed that the replacement for the Arg residue reduced the binding free energies of E-F sites and the rate constant of transglycosylation. The rate constant of transglycosylation for R114A was about half of that for the wildtype enzyme. H-1-NMR analysis of R114H and R114A indicated that the structural changes induced by the mutations were not restricted to the region surrounding Arg114,, but rather extended to the aromatic side chains of Phe34 and Trp123, of which the signals are connected with each other through nuclear Overhauser effect (NOE) in the wild-type. We speculate that such a conformational change causes differences in substrate and acceptor binding at subsites E and F, lowering the efficiency of glycosyl transfer reaction of lysozyme.