title: Site-directed mutagenesis of arginine 179 of thymidylate synthase. A nonessential substrate-binding residue.
abstract: X-ray structural studies have shown that Arg-179 of thymidylate synthase is complexed to bound inorganic phosphate or to the 5'-phosphate of the bound substrate dUMP. The importance of Arg-179 to the structure/function of thymidylate synthase is also indicated by its complete conservation among the 17 thymidylate synthases thus far sequenced. In the present work, Arg-179 has been replaced by Thr, Ala, Lys, and Glu using site-directed mutagenesis with a mixture of four synthetic oligonucleotides as primers. The mutant proteins complement thymidylate synthase-deficient Escherichia coli and show high enzyme activity. Each of these mutants has been purified to homogeneity, partially sequenced to verify the mutation, and has had its steady state kinetic parameters determined. The most significant effect of all mutations is localized to a decrease in the net rate of association of thymidylate synthase with dUMP; the Lys mutant also shows an apparent increase in the dissociation constant of the folate cofactor of the reaction. The high activity in the mutant enzymes is explained by "plasticity" of the enzyme and compensatory actions of the other Arg residues. Why the Arg-179 residue has been conserved during evolution remains an open question.
authors.affiliation: Department of Biochemistry, University of California, San Francisco 94143.
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
authors.forename: K
authors.initials: K
authors.lastname: Pinter
authors.forename: J
authors.initials: J
authors.lastname: Kealey
authors.forename: V J
authors.initials: VJ
authors.lastname: Davisson
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D013940:Thymidylate Synthase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D001120:Arginine; D013940:Thymidylate Synthase