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.
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Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Suhadolnik
R J
RJ
D015088:2',5'-Oligoadenylate Synthetase / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D000345:Affinity Labels; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D010777:Photochemistry; D011070:Poly I-C / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D000345:Affinity Labels; D001386:Azides; D017931:DNA Primers; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D015088:2',5'-Oligoadenylate Synthetase; D011070:Poly I-C