Howell E E, Villafranca J E, Warren M S, Oatley S J, Kraut J
Science. 1986 Mar 7;231(4742):1123-8. doi: 10.1126/science.3511529.
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.
为了阐明底物结合位点处一个不变的羧酸（27位的天冬氨酸）的功能作用，对两种突变型二氢叶酸还原酶（大肠杆菌）的晶体结构和酶学性质进行了研究。通过寡核苷酸定向诱变构建的一种突变，用天冬酰胺取代了27位的天冬氨酸；另一种是27位回复为丝氨酸的原位回复突变体。唯一的结构扰动涉及两个内部结合的水分子。两种突变体都具有低但易于测量的活性，该活性随pH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。得出的结论是，27位的天冬氨酸参与底物的质子化，但不参与带正电荷的质子化过渡态的静电稳定。
