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Science 18 November 1983:
Vol. 222. no. 4625, pp. 782 - 788
DOI: 10.1126/science.6356360
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Articles

Science, Vol 222, Issue 4625, 782-788
Copyright © 1983 by American Association for the Advancement of Science

articles

Directed mutagenesis of dihydrofolate reductase

JE Villafranca, EE Howell, DH Voet, MS Strobel, RC Ogden, JN Abelson, and J Kraut

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.


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