ENZYMATIC-ACTIVITY OF 2'-5'-OLIGOADENYLATE SYNTHETASE IS IMPAIRED BY SPECIFIC MUTATIONS THAT AFFECT OLIGOMERIZATION OF THE PROTEIN

Previous studies from our laboratory have shown that deletion of resid ues 321 to 344 of the 9-2 isozyme of 2'-5'-oligoadenylate (2-5(A)) syn thetase causes a loss of its enzyme activity (Ghosh, S. K., Kusari, J. , Bandyopadhyay, S. K., Samanta, H., Kumar, R., and Sen, G. C. (1991) J. Biol. Chem. 266, 15293-15299). Sequence comparison of this region a mong the different isozymes of 2-5(A) synthetases revealed that the re sidues at positions 330 to 333 are highly conserved. Alanine-scanning mutagenesis of these residues demonstrated that the residues present a t 331, 332, and 333 are important for activity but the proline at posi tion 330 was dispensable. The triple mutant containing Ala residues at 331, 332, and 333 was completely inactive. Different double mutants w ere slightly active, and the three single mutants were partially activ e. The triple mutant was further characterized for delineating the nat ure of its defect. The mutant protein was enzymatically inactive irres pective of whether it was synthesized in rabbit reticulocyte lysate, E scherichia coli or Trichoplusia ni insect cells. It could bind double- stranded RNA and ATP as efficiently as the wild type protein. It was, however, defective in oligomerization. Gel filtration and sedimentatio n velocity analyses of in vitro synthesized proteins revealed that the wild type protein, but not the triple mutant, formed tetramers. The t etrameric fraction, but not the monomeric fraction of the wild type pr otein was enzymatically active. The failure of the triple mutant to pa rticipate in homomeric protein-protein interaction was confirmed by in vivo assays in insect cells. These results indicate that tetramerizat ion of the protein is required for the enzymatic activity of the small 2-5(A) synthetases.