title: Complete replacement set of amino acids at the C-terminus of thymidylate synthase: quantitative structure-activity relationship of mutants of an enzyme.
abstract: The C-terminal residue of thymidylate synthase (TS) is highly conserved and has been implicated in cofactor binding, catalysis, and a conformational change. The codon for the C-terminal valine of Lactobacillus casei TS has been replaced with those for 19 other amino acids and the amber stop codon. Fourteen of the resulting mutant proteins were active by genetic complementation using a Thy- strain of Escherichia coli, and 18 mutants were active by in vitro assay. Only the aspartate and amber mutations had undetectable activity. All of the mutants were expressed at high levels (5-30% of soluble protein) and were purified by phosphocellulose chromatography. In general, the alterations at position 316 led to little effect on the Km for dUMP, an increase in Km for the folate cofactor, and a decrease in kcat. The observations show that TS can tolerate the substitution of most amino acids for valine at the C-terminus without a complete loss of activity, that hydrophobic substitutions are preferred, and that the C-terminal side chain is involved in both cofactor binding and catalysis. There was an excellent correlation between log kcat and hydrophobicity of the side chain at position 316 and an inverse correlation between log Km and the hydrophobicity of this residue. Kinetic parameters of the cofactor-independent TS-catalyzed dehalogenation of BrdUMP showed no variation with the side chain at position 316. In context of the structure of TS, it is proposed that binding of the cofactor triggers a conformational change in which the C-terminal side chain undergoes hydrophobic interactions that stabilize a rate-limiting transition state of the TS reaction.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
authors.forename: S C
authors.initials: SC
authors.lastname: Climie
authors.forename: C W
authors.initials: CW
authors.lastname: Carreras
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D003062:Codon; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014633:Valine / Q000235:genetics
chemical_list: D003062:Codon; D003856:Deoxyuracil Nucleotides; C017089:bromodeoxyuridylate; D013940:Thymidylate Synthase; D014633:Valine