Amin N, Peterkofsky A
Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, Bethesda, MD 20892.
Biochem Biophys Res Commun. 1992 Feb 14;182(3):1218-25. doi: 10.1016/0006-291x(92)91861-j.
The region of Escherichia coli adenylyl cyclase spanned by glycine-55 to threonine-65 was tested for its importance for enzyme activity. Site-directed mutagenesis was used to replace glycine-55 and glycine-60 as well as lysine-59, leucine-63 and threonine-65 with other amino acids. While substitution of glycine-55 with aspartic acid produced no significant change in kinetic parameters, the change of glycine-60 to aspartic acid or asparagine eliminated binding to 8-azido-ATP and decreased the Vmax (two orders of magnitude) and Km (factor of four-five). Smaller effects on kinetic parameters were observed with substitutions of lysine-59, leucine-63 or threonine-65.
对大肠杆菌腺苷酸环化酶中甘氨酸55至苏氨酸65所跨越的区域进行了酶活性重要性测试。采用定点诱变将甘氨酸55、甘氨酸60以及赖氨酸59、亮氨酸63和苏氨酸65替换为其他氨基酸。虽然用天冬氨酸替代甘氨酸55在动力学参数上未产生显著变化，但将甘氨酸60变为天冬氨酸或天冬酰胺会消除与8-叠氮基ATP的结合，并降低Vmax（两个数量级）和Km（四五倍因子）。用赖氨酸59、亮氨酸63或苏氨酸65进行替换时，观察到对动力学参数的影响较小。
