Effects of specific mutations in active site motifs of 2 ',5 '-oligoadenylate synthetase on enzymatic activity

2',5'-Olipoadenglate synthetase (2',5'-OAS) is a double-stranded RNA-depend ent nucleotidyl-transferase induced by interferon (IFN). Several 2',5'-GAS cDN4 have been cloned from human, pig, rat. mouse, and chicken. a P-loop mo tif followed by an Asp-containing sequence (referred to as D-box) and a reg ion with a high content of Lys and Arg (KR-rich region) are well conserved in 2',5'-GAS, The sequence (196)DFLKQR(201) of 40-kDa human 2',5'-GAS, to w hich 8-azido-ATP binds (N, Kon and R.J. Suhadolnik, J, Biol, Chem. 271, 199 83-19990, 1996), is included in the KR-rich region. We introduced several s ite-directed mutations into these active motifs of 42-kDa murine 2',5'-GAS. Each mutant enzyme studied bound to poly(I):poly(C) to the same extent as wild-type enzyme. Both K67R, a P-loop mutant, and K200R, a KR-rich region m utant, exhibited a reduced but considerable rate of enzymatic activities, T he activity of the double mutant K67R/K200R was about 10% of the wild type. On the other hand, the activities of both K67M and K200M were not more tha n 2% of the wild-type enzyme, and no activity was detected in another P-loo p mutant, G62A/G63A, The binding of Mg2+ to a D-box mutant D76N/D78N was ma rkedly reduced, and only a very low level of enzymatic activity was detecte d in this mutant. These results demonstrate that the P-loop, the D-box that binds Mg2+, and the KR-rich region are important for the enzymatic activit ies of 2',5'-GAS.