The nature of the catalytic domain of 2 '-5 '-oligoadenylate synthetases

2'-5'-Oligoadenylate (2-5(A)) synthetases are a family of interferon-induce d enzymes that are activated by double-stranded RNA. To understand why, unl ike other DNA and RNA polymerases, they catalyze 2'-5' instead of 3'-5' pho sphodiester bond formation, we used molecular modeling to compare the struc ture of the catalytic domain of DNA polymerase beta (pol beta) to that of a region of the P69 isozyme of 2-5(A) synthetase. Although the primary seque nce identity is low, like pol beta, P69 can assume an alpha beta beta alpha beta beta beta structure in this region. Moreover, mutation of the three A sp residues of P69, which correspond to the three catalytic site Asp residu es of pol beta, inactivated the enzyme without affecting its substrate and activator binding capacity, providing further credence to the concept that this region is the catalytic domain of P69. This domain is highly conserved among all 2-5(A) synthetase isozymes, Biochemical and mutational studies d emonstrated that dimerization of the P69 protein is required for its enzyme activity. However, a dimer containing a wild type subunit and an inactive catalytic domain mutant subunit was also active. The rate of catalysis of t he heterodimer was half of that of the wild type homodimer, although the tw o proteins bound double-stranded RNA and ATP equally well.