title: Contribution of a salt bridge to binding affinity and dUMP orientation to catalytic rate: mutation of a substrate-binding arginine in thymidylate synthase.
abstract: Invariant arginine 179, one of four arginines that are conserved in all thymidylate synthases (TS) and that bind the phosphate moiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP), can be altered even to a negatively charged glutamic acid with little effect on kcat. In the mutant structures, ordered water or the other phosphate-binding arginines compensate for the hydrogen bonds made by Arg179 in the wild-type enzyme and there is almost no change in the conformation or binding site of dUMP. Correlation of dUMP Kds for TS R179A and TS R179K with the structures of their binary complexes shows, that the positive charge on Arg179 contributes significantly to dUMP binding affinity. kcat/K(m) for dUMP measures the rate of dUMP binding to TS during the ordered bi-substrate reaction, and in the ternary complex dUMP provides a binding surface for the cofactor. kcat/K(m) reflects the ability of the enzyme to accept a properly oriented dUMP for catalysis and is less sensitive than is Kd to the changes in electrostatics at the phosphate binding site.
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco 94143-0448, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Finer-Moore
authors.forename: E B
authors.initials: EB
authors.lastname: Fauman
authors.forename: R J
authors.initials: RJ
authors.lastname: Morse
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
authors.forename: R M
authors.initials: RM
authors.lastname: Stroud
mesh_terms: D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008958:Models, Molecular; D015394:Molecular Structure; D016296:Mutagenesis; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D003856:Deoxyuracil Nucleotides; D010710:Phosphates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D001120:Arginine; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase