Villafranca J E, Howell E E, Voet D H, Strobel M S, Ogden R C, Abelson J N, Kraut J
Science. 1983 Nov 18;222(4625):782-8. doi: 10.1126/science.6356360.
Three mutations of the enzyme dihydrofolate reductase were constructed by oligonucleotide-directed mutagenesis of the cloned Escherichia coli gene. The mutations--at residue 27, aspartic acid replaced with asparagine; at residue 39, proline replaced with cysteine; and at residue 95, glycine replaced with alanine--were designed to answer questions about the relations between molecular structure and function that were raised by the x-ray crystal structures. Properties of the mutant proteins show that Asp-27 is important for catalysis and that perturbation of the local structure at a conserved cis peptide bond following Gly-95 abolishes activity. Substitution of cysteine for proline at residue 39 results in the appearance of new forms of the enzyme that correspond to various oxidation states of the cysteine. One of these forms probably represents a species cross-linked by an intrachain disulfide bridge between the cysteine at position 85 and the new cysteine at position 39.
通过对克隆的大肠杆菌基因进行寡核苷酸定向诱变，构建了二氢叶酸还原酶的三种突变体。这些突变分别是：第27位残基处，天冬氨酸被天冬酰胺取代；第39位残基处，脯氨酸被半胱氨酸取代；第95位残基处，甘氨酸被丙氨酸取代。这些突变旨在回答由X射线晶体结构提出的关于分子结构与功能之间关系的问题。突变蛋白的特性表明，Asp-27对催化作用很重要，并且Gly-95之后保守的顺式肽键处局部结构的扰动会使活性丧失。在第39位残基处用半胱氨酸取代脯氨酸会导致酶出现新的形式，这些新形式对应于半胱氨酸的各种氧化态。其中一种形式可能代表一种由第85位的半胱氨酸与第39位的新半胱氨酸之间的链内二硫键交联的物种。
