Identification of the ATP binding domain of recombinant human 40-kDa 2',5'-oligoadenylate synthetase by photoaffinity labeling with 8-azido-[alpha-32P]ATP.

Three isoforms of the interferon-inducible 2',5'-oligoadenylate (2-5A) synthetase that require double-stranded RNA have been isolated and cloned. However, identification of the amino acid(s) of 2-5A synthetase directly interacting with ATP is crucial to the elucidation of the mechanism of the enzymatic conversion of ATP to 2',5'-oligoadenylates by 2-5A synthetase. Recombinant human 40-kDa 2-5A synthetase has been expressed as a glutathione S-transferase fusion protein in E. coli and purified to near homogeneity in milligram quantities. The azido photoprobe, 8-azido-[alpha-32P]ATP, has been used to identify the ATP binding domain of the recombinant human 40-kDa 2-5A synthetase. Specific covalent photoincorporation of 8-azido-[alpha-32P]ATP into the 2-5A synthetase, tryptic digestion of the covalently 32P-labeled enzyme, isolation of the photolabeled phosphopeptide by metal (Al3+) chelate chromatography, and high pressure liquid chromatography identified a 32P-pentapeptide, which has been assigned to the ATP binding domain of 2-5A synthetase. The radioactive pentapeptide has the sequence D196FLKQ200 in which the photoprobe, 8-azido-[alpha-32P]ATP, chemically modified the amino acid lysine 199. The catalytic importance of Lys199 was further established by mutation of lysine 199 to arginine 199 and histidine 199 using site-directed mutagenesis. The K199R and K199H recombinant human 40-kDa 2-5A synthetase mutants bind 8-azido-ATP and the allosteric activator, poly(I) poly(C) but are enzymatically inactive. These photoaffinity labeling and mutation data strongly suggest that lysine 199 is essential for the formation of a productive 2-5A synthetase-ATP-double-stranded RNA complex for the enzymatic conversion of ATP to 2-5A.