Inoue M, Yamada H, Yasukochi T, Miki T, Horiuchi T, Imoto T
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Biochemistry. 1992 Oct 27;31(42):10322-30. doi: 10.1021/bi00157a021.
The "right-sided" and "left-sided" substrate binding modes at the lower saccharide binding subsites (D-F sites) of chicken lysozyme were investigated by utilizing mutant lysozymes secreted from yeast. We constructed the following mutant lysozymes; "left-sided" substitution of Asn46 to Asp, deletion of Thr47, and insertion of Gly between Thr47 and Asp48 and "right-sided" substitution of Asn37 to Gly. Analyses of their activities and substrate binding abilities showed that Asn46 and Thr47 are involved in the initial enzyme-substrate complex and Asn37 is involved in the transition state. These results support an earlier proposal that interactions between substrate and residues at the left side of lysozyme stabilize a catalytically inactive enzyme-substrate complex, while interactions between substrate and residues at the right side stabilize the catalytically active complex [Pincus, M. R., & Scheraga, H. A. (1979) Macromolecules 12, 633-644]. These results are also consistent with the proposed kinetic mechanism for lysozyme reaction that the rearrangement of an initial enzyme-substrate complex (beta-complex) to another complex (gamma-complex) is required for catalytic hydrolysis [Banerjee S. K., Holler, E., Hess, G. P., & Rupley, J. A. (1975) J. Biol. Chem. 250, 4355-4367].
利用酵母分泌的突变型溶菌酶，研究了鸡溶菌酶在较低糖结合亚位点（D - F位点）的“右侧”和“左侧”底物结合模式。我们构建了以下突变型溶菌酶：将Asn46替换为Asp的“左侧”突变、删除Thr47以及在Thr47和Asp48之间插入Gly，还有将Asn37替换为Gly的“右侧”突变。对它们的活性和底物结合能力的分析表明，Asn46和Thr47参与初始酶 - 底物复合物的形成，而Asn37参与过渡态的形成。这些结果支持了一个早期的提议，即溶菌酶左侧的底物与残基之间的相互作用稳定了催化无活性的酶 - 底物复合物，而右侧的底物与残基之间的相互作用稳定了催化活性复合物[平卡斯，M. R.，& 谢拉加，H. A.（1979年）《大分子》12卷，633 - 644页]。这些结果也与所提出的溶菌酶反应动力学机制一致，即催化水解需要将初始酶 - 底物复合物（β - 复合物）重排为另一种复合物（γ - 复合物）[班纳吉，S. K., 霍勒，E., 赫斯，G. P., & 鲁普利，J. A.（1975年）《生物化学杂志》250卷，4355 - 4367页]。
