Malcolm B A, Rosenberg S, Corey M J, Allen J S, de Baetselier A, Kirsch J F
Department of Biochemistry, University of California, Berkeley.
Proc Natl Acad Sci U S A. 1989 Jan;86(1):133-7. doi: 10.1073/pnas.86.1.133.
The roles of the catalytic active-site residues aspartic acid-52 and glutamic acid-35 of chicken lysozyme (EC 3.2.1.17) have been investigated by separate in vitro mutagenesis of each residue to its corresponding amide (denoted as D52N and E35Q, respectively). The mutant enzyme D52N exhibits approximately 5% of the wild-type lytic activity against Micrococcus luteus cell walls, while there is no measurable activity associated with E35Q (0.1% +/- 0.1%). The measured dissociation constants for the chitotriose-enzyme complexes were 4.1 microM (D52N) and 13.4 microM (E35Q) vs. 8.6 microM for wild type, indicating that the alterations in catalytic properties may be due in part to binding effects as well as to direct catalytic participation of these residues. The mutant lysozymes have been expressed in and secreted from yeast and obtained at a level of approximately 5 mg per liter of culture by high-salt elution from the cell walls.
通过将鸡溶菌酶（EC 3.2.1.17）的催化活性位点残基天冬氨酸-52和谷氨酸-35分别体外突变为其相应的酰胺（分别记为D52N和E35Q），对它们的作用进行了研究。突变酶D52N对藤黄微球菌细胞壁表现出约5%的野生型裂解活性，而E35Q没有可测量的活性（0.1%±0.1%）。测得的壳三糖 - 酶复合物的解离常数，野生型为8.6微摩尔，D52N为4.1微摩尔，E35Q为13.4微摩尔，这表明催化特性的改变可能部分归因于结合效应以及这些残基的直接催化参与。突变溶菌酶已在酵母中表达并分泌，通过从细胞壁上进行高盐洗脱，每升培养物可获得约5毫克的产量。
