FUNCTIONAL-ROLE OF ASPARTIC ACID-27 IN DIHYDROFOLATE-REDUCTASE REVEALED BY MUTAGENESIS
FUNCTIONAL-ROLE OF ASPARTIC ACID-27 IN DIHYDROFOLATE-REDUCTASE REVEALED BY MUTAGENESIS
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DOI：
10.1126/science.3511529
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发表时间：
1986-03-07
期刊：
影响因子：
56.9
通讯作者：
KRAUT, J
中科院分区：
文献类型：
--
作者：
HOWELL, EE;VILLAFRANCA, JE;KRAUT, J
研究了两种突变二氢叶酸还原酶（大肠杆菌）的晶体结构和酶性质，以阐明底物结合位点上不变的羧酸（第 27 位天冬氨酸）的功能作用。一种通过寡核苷酸定向诱变构建的突变，用天冬酰胺取代了 Asp27；另一个是 Ser27 的主位点回复。唯一的结构扰动涉及两个内部结合的水分子。两种突变体都具有较低但易于测量的活性，并且随着 pH 值的降低而迅速增加。还对突变酶的相对叶酸：二氢叶酸活性和动力学氘同位素效应进行了表征。结论是，Asp27 参与底物的质子化，但不参与带正电的质子化过渡态的静电稳定。
The crystal structures and enzymic properties of two mutant dihydrofolate reductases (Escherichia coli) were studied in order to clarify the functional role of an invariant carboxylic acid (aspartic acid at position 27) at the substrate binding site. One mutation, constructed by oligonucleotide-directed mutagenesis, replaces Asp27 with asparagine; the other is a primary-site revertant to Ser27. The only structural perturbations involve two internally bound water molecules. Both mutants have low but readily measurable activity, which increases rapidly with decreasing pH. The mutant enzymes were also characterized with respect to relative folate: dihydrofolate activities and kinetic deuterium isotope effects. It is concluded that Asp27 participates in protonation of the substrate but not in electrostatic stabilization of a positively charged, protonated transition state.