Drabikowska A K, Woźniak G
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa.
Biochem J. 1990 Sep 1;270(2):319-23. doi: 10.1042/bj2700319.
Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.
来自大肠杆菌的尿苷磷酸化酶在pH 7.1和10℃下被焦碳酸二乙酯灭活，二级反应速率常数为840 M-1·min-1。灭活速率随pH升高而增加，表明存在一个pK为6.6的氨基酸残基参与其中。添加到失活酶中的羟胺可恢复其活性。每个酶亚基有三个组氨酸残基被焦碳酸二乙酯修饰。对残余酶活性以及修饰组氨酸残基数量的动力学和统计分析表明，在这三个可修饰残基中，只有一个对酶活性至关重要。这个组氨酸残基的反应活性比其他两个残基高出10倍以上。尿苷虽然浓度很高，但可保护酶不被灭活，并保护这个反应活性很强的组氨酸残基不被修饰。因此可以得出结论，尿苷磷酸化酶的六个亚基中每个亚基仅含有一个对酶活性至关重要的组氨酸残基。
