Identification of the substrate-binding sites of 2'-5'-oligoadenylate synthetase.
2'-5'-Oligoadenylate synthetases are interferon-induced enzymes that upon activation by double-stranded RNA polymerize ATP to 2'-5'-linked oligoadenylates. In our continuing effort to understand the mechanism of catalysis by these enzymes, we used photo affinity cross-linking and peptide mapping to identify the substrate-binding sites of the P69 isozyme of human 2'-5'-oligoadenylate synthetases. Radiolabeled azido 2'-5'-oligoadenylate dimers were enzymatically synthesized and used as ligands for cross-linking to the P69 protein by exposure to ultraviolet light. The radiolabeled protein was digested with trypsin, and two ligand-cross-linked peptides were purified by immobilized aluminum affinity chromatography followed by reverse phase high pressure liquid chromatography. The peptides were identified by mass spectrometry and peptide sequencing and were found to correspond to residues 420-425 and 539-547 of P69. To examine the functional importance of the cross-linking sites, specific residues in the two peptides were mutated. When residues in the two sites were mutated individually, ligand cross-linking was selectively eliminated at the mutated site, and the enzyme activity was lost almost completely. Using substrates that can serve either as a donor or as an acceptor but not both, we could identify one of the sites as the acceptor and the other as the donor site.
Sarkar
Saumendra N
SN
Department of Molecular Biology, The Lerner Research Institute and the Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Miyagi
Masaru
M
Crabb
John W
JW
Sen
Ganes C
GC
D015088:2',5'-Oligoadenylate Synthetase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D020134:Catalytic Domain; D019281:Dimerization; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
D007527:Isoenzymes; D008024:Ligands; D010446:Peptide Fragments; D015088:2',5'-Oligoadenylate Synthetase