IDENTIFICATION OF THE ATP BINDING DOMAIN OF RECOMBINANT HUMAN 40-KDA 2',5'-OLIGOADENYLATE SYNTHETASE BY PHOTOAFFINITY-LABELING WITH 8-AZIDO-[ALPHA-P-32]ATP

Three isoforms of the interferon-inducible 2',5'-oligoadenylate (2-5A) synthetase that require double-stranded RNA have been isolated and cl oned. However, identification of the amino acid(s) of 2-5A synthetase directly interacting with ATP is crucial to the elucidation of the mec hanism of the enzymatic conversion of ATP to 2',5'-oligoadenylates by 2-5A synthetase. Recombinant human 40-kDa 2-5A synthetase has been exp ressed as a glutathione S-transferase fusion protein in E. coli and pu rified to near homogeneity in milligram quantities. The azido photopro be, 8-azido-[alpha-P-32]ATP, has been used to identify the ATP binding domain of the recombinant human 40-kDa 2-5A synthetase. Specific cova lent photoincorporation of 8-azido-[alpha-P-32]ATP into the 2-5A synth etase, tryptic digestion of the covalently P-32-labeled enzyme, isolat ion of the photolabeled phosphopeptide by metal (Al3+) chelate chromat ography, and high pressure liquid chromatography identified a P-32-pen tapeptide, which has been assigned to the ATP binding domain of 2-5A s ynthetase. The radioactive pentapeptide has the sequence D(196)FLKQ(20 0) in which the photoprobe, 8-azido-[alpha-P-32]ATP, chemically modifi ed the amino acid lysine 199. The catalytic importance of Lys(199) was further established by mutation of lysine 199 to arginine 199 and his tidine 199 using site directed mutagenesis, The K199R and K199H recomb inant human 40-kDa 2-5A synthetase mutants bind 8-azido-ATP and the al losteric activator, poly(I). poly(C) but are enzymatically inactive. T hese photoaffinity labeling and mutation data strongly suggest that ly sine 199 is essential for the formation of a productive 2-5A synthetas e-ATP double-stranded RNA complex for the enzymatic conversion of ATP to 2-5A.