title: Site-directed mutagenesis and deletion of the carboxyl terminus of Escherichia coli ribonucleotide reductase protein R2. Effects on catalytic activity and subunit interaction.
abstract: Ribonucleotide reductase from Escherichia coli consists of two dissociable, nonidentical homodimeric proteins called R1 and R2. The role of the C-terminal region of R2 in forming the R1R2 active complex has been studied. A heterodimeric R2 form with a full-length polypeptide chain and a truncated one missing the last 30 carboxyl-terminal residues was engineered by site-directed mutagenesis. Kinetic analysis of the binding of this protein to R1, compared with full-length or truncated homodimer, revealed that the C-terminal end of R2 accounts for all of its interactions with R1. The intrinsic dissociation constant of the heterodimeric R2 form, with only one contact to R1, 13 microM, is of the same magnitude as that obtained previously [Climent, I., Sjöberg, B.-M., & Huang, C. Y. (1991) Biochemistry 30, 5164-5171] for synthetic C-terminal peptides, 15-18 microM. We have also mutagenized the only two invariant residues localized at the C-terminal region of R2, glutamic acid-350 and tyrosine-356, to alanine. The binding of these mutant proteins to R1 remains tight, but their catalytic activity is severely affected. While E350A protein exhibits a low (240 times less active than the wild-type) but definitive activity, Y356A is completely inactive. A catalytic rather than structural role for these residues is discussed.
authors.affiliation: Department of Molecular Biology, Stockholm University, Sweden.
authors.forename: I
authors.initials: I
authors.lastname: Climent
authors.forename: B M
authors.initials: BM
authors.lastname: Sjöberg
authors.forename: C Y
authors.initials: CY
authors.lastname: Huang
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002384:Catalysis; D002499:Centrifugation, Density Gradient; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000494:pharmacology; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases