Kawamura Shunsuke, Chijiiwa Yuki, Minematsu Toshie, Fukamizo Tamo, Vårum Kjell M, Torikata Takao
Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto 869-1404, Japan.
Biosci Biotechnol Biochem. 2008 Mar;72(3):823-32. doi: 10.1271/bbb.70694. Epub 2008 Mar 7.
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 wild-type enzyme. (1)H-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.
为了更好地理解亚位点E和F在溶菌酶催化反应中的作用，制备了突变酶，其中位于鸡蛋清溶菌酶（HEL）亚位点E和F右侧的精氨酸114被赖氨酸、组氨酸或丙氨酸取代。用组氨酸或丙氨酸取代精氨酸114会降低对人工底物几丁质糖的水解活性，而用赖氨酸取代则影响不大。用底物N-乙酰葡糖胺五聚体（GlcNAc）5进行的动力学分析表明，精氨酸残基的取代降低了E-F位点的结合自由能和转糖基化速率常数。R114A的转糖基化速率常数约为野生型酶的一半。对R114H和R114A的（1）H-NMR分析表明，突变引起的结构变化不仅限于精氨酸114周围区域，而是延伸到苯丙氨酸34和色氨酸123的芳香侧链，在野生型中，它们的信号通过核Overhauser效应（NOE）相互连接。我们推测这种构象变化会导致亚位点E和F处底物和受体结合的差异，从而降低溶菌酶糖基转移反应的效率。
