Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity.
Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity.
复制标题
改变大肠杆菌碱性磷酸酶活性位点的天冬氨酸 101 可增强催化活性。
DOI：
10.1093/protein/3.2.127
复制
发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Kantrowitz,ER
中科院分区：
文献类型：
--
作者：
Chaidaroglou,A;Kantrowitz,ER
The function of aspartic acid residue 101 in the active site ofEscherichia colialkaline phosphatase was investigated by site-specific mutagenesis. A mutant version of alkaline phosphatase was constructed with alanine in place of aspartic acid at position 101. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, pH 8.0, both thekcatand theKm, for the mutant enzyme increase by –2-fold, resulting in almost no change in thekcat/Kmratio. Under conditions of no external phosphate acceptor and pH 8.0, both thekcatand theKmfor the mutant enzyme decrease by ˜2-fold, again resulting in almost no change in thekcat/Kmratio. Thekcatfor the hydrolysis of 4-methyl-umbelliferyl phosphate andp-nitrophenyl phosphate are nearly identical for both the wild-type and mutant enzymes, as is theK1for inorganic phosphate. The replacement of aspartic acid 101 by alanine does have a significant effect on the activity of the enzyme as a function of pH, especially in the presence of a phosphate acceptor. At pH 9.4 the mutant enzyme exhibits 3-fold higher activity than the wild-type. The mutant enzyme also exhibits a substantial decrease in thermal stability: it is half inactivated by treatment at 49°C for 15 min compared to 71°C for the wild-type enzyme. The data reported here suggest that this amino acid substitution alters the rates of steps after the formation of the phospho-enzyme intermediate. Analysis of the X-ray structure of the wild-type enzyme indicates that the increase in catalytic rate of the mutant enzyme in the presence of a phosphate acceptor may be due to an increase in accessibility of the active site near Serl02. The increased catalytic rate of this mutant enzyme may be utilized to improve diagnostic tests that require alkaline phosphatase, and the reduced heat stability of the mutant enzyme may make it useful in recombinant DNA techniques that require the ability to heat-inactivate the enzyme after use.
