SITE-DIRECTED MUTAGENESIS AND DELETION OF THE CARBOXYL TERMINUS OF ESCHERICHIA-COLI RIBONUCLEOTIDE REDUCTASE PROTEIN R2 - EFFECTS ON CATALYTIC ACTIVITY AND SUBUNIT INTERACTION

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-mu-M, is of the same magnitude as that obtained previously [Climent, I., Sjoberg, B.-M., & Huang, C. Y. (1991) Biochemistry 30, 5164-5171] for synthetic C-terminal peptides, 15-18-mu-M. 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.