Kumagai I, Maenaka K, Sunada F, Takeda S, Miura K
Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo, Bunkyo-ku, Japan.
Eur J Biochem. 1993 Feb 15;212(1):151-6. doi: 10.1111/j.1432-1033.1993.tb17645.x.
The subsite structures in the active site of hen egg-white lysozyme were altered by site-directed mutagenesis. Replacement of Trp62, which is involved in apolar interaction with a sugar ring, and Asp101, which is hydrogen bonded to the same sugar ring in subsite B, led to a shift of the oligosaccharide-binding mode in the active-site cleft. Consequently, the double-mutant lysozyme (Trp62His, Asp101Gly) exhibited a drastic change of substrate-binding without any significant loss of enzymic activity. Conversion of Asn37, which is postulated to be involved in interaction with a sugar ring in subsite F, had a reverse effect on substrate binding. Nuclear magnetic resonance analysis of mutant lysozymes, in which Trp62 was replaced with Phe or His, suggested that these replacements not only altered the structure of the amino acid chain at position 62 of the lysozyme, but also induced local structural changes around the residue at position 62.
通过定点诱变改变了鸡蛋清溶菌酶活性位点中的亚位点结构。参与与糖环进行非极性相互作用的色氨酸62以及在亚位点B中与同一糖环形成氢键的天冬氨酸101被替换，导致活性位点裂隙中寡糖结合模式发生转变。因此，双突变溶菌酶（色氨酸62突变为组氨酸，天冬氨酸101突变为甘氨酸）表现出底物结合的剧烈变化，而酶活性没有任何显著损失。据推测参与与亚位点F中的糖环相互作用的天冬酰胺37的转化对底物结合产生了相反的影响。对色氨酸62被苯丙氨酸或组氨酸替换的突变溶菌酶进行核磁共振分析表明，这些替换不仅改变了溶菌酶第62位氨基酸链的结构，还诱导了第62位残基周围的局部结构变化。
