Muraki M, Goda S, Nagahora H, Harata K
Biomolecules Department, National Institute of Bioscience and Human-Technology, Ibaraki, Japan.
Protein Sci. 1997 Feb;6(2):473-6. doi: 10.1002/pro.5560060227.
The importance of van der Waals contact between Glu 35 and Trp 109 to the active-site structure and the catalytic properties of human lysozyme (HL) has been investigated by site-directed mutagenesis. The X-ray analysis of mutant HLs revealed that both the replacement of Glu 35 by Asp or Ala, and the replacement of Trp 109 by Phe or Ala resulted in a significant but localized change in the active-site cleft geometry. A prominent movement of the backbone structure was detected in the region of residues 110 to 120 and in the region of residues 100 to 115 for the mutations concerning Glu 35 and Trp 109, respectively. Accompanied by the displacement of the main-chain atoms with a maximal deviation of C alpha atom position ranging from 0.7 A to 1.0 A, the mutant HLs showed a remarkable change in the catalytic properties against Micrococcus luteus cell substrate as compared with native HL. Although the replacement of Glu 35 by Ala completely abolished the lytic activity, HL-Asp 35 mutant retained a weak but a certain lytic activity, showing the possible involvement of the side-chain carboxylate group of Asp 35 in the catalytic action. The kinetic consequence derived from the replacement of Trp 109 by Phe or Ala together with the result of the structural change suggested that the structural detail of the cleft lobe composed of the residues 100 to 115 centered at Ala 108 was responsible for the turnover in the reaction of HL against the bacterial cell wall substrate. The results revealed that the van der Waals contact between Glu 35 and Trp 109 was an essential determinant in the catalytic action of HL.
通过定点诱变研究了人溶菌酶（HL）中谷氨酸35（Glu 35）和色氨酸109（Trp 109）之间的范德华接触对活性位点结构和催化特性的重要性。突变型HL的X射线分析表明，用天冬氨酸（Asp）或丙氨酸（Ala）取代Glu 35，以及用苯丙氨酸（Phe）或Ala取代Trp 109，都会导致活性位点裂隙几何形状发生显著但局部的变化。对于涉及Glu 35和Trp 109的突变，分别在残基110至120区域和残基100至115区域检测到主链结构的显著移动。伴随着主链原子的位移，Cα原子位置的最大偏差范围为0.7埃至1.0埃，与天然HL相比，突变型HL对藤黄微球菌细胞底物的催化特性发生了显著变化。虽然用Ala取代Glu 35完全消除了裂解活性，但HL-Asp 35突变体保留了微弱但一定的裂解活性，表明Asp 35的侧链羧基可能参与了催化作用。用Phe或Ala取代Trp 109所产生的动力学结果以及结构变化的结果表明，以Ala 108为中心的由残基100至115组成的裂隙叶的结构细节是HL与细菌细胞壁底物反应中周转的原因。结果表明，Glu 35和Trp 109之间的范德华接触是HL催化作用的一个重要决定因素。
