Lipford J R, Worland S T, Farnet C M
Dana-Farber Cancer Institute, Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115.
J Acquir Immune Defic Syndr (1988). 1994 Dec;7(12):1215-23.
Recombinant human immunodeficiency virus type 1 (HIV-1) integrase was shown to bind ATP and other nucleoside triphosphates and nucleotide analogs in vitro. Cross-linking of ATP and the photoaffinity analog 8-azido-ATP to integrase occurred in a UV dose-dependent manner. Covalent binding of ATP to integrase was also achieved without UV irradiation when the nucleotide was oxidized to the 2',3'-dialdehyde derivative (oxidized ATP) prior to incubation with the protein, indicating the presence of a reactive lysine residue in the nucleotide binding region of the protein. A number of experimental observations indicate that nucleotides and DNA substrates bind at the same or overlapping site(s) on the integrase protein. For example, the binding of nucleotides or nucleotide analogs to integrase was blocked by prior incubation with DNA substrates, and the covalent cross-linking of 8-azido-ATP to integrase inhibited the DNA binding and oligonucleotide cleavage activities of the protein. Oxidized ATP inhibited the oligonucleotide cleavage activity of integrase at concentrations that had no effect on DNA binding, suggesting that oxidized nucleotides may specifically target the catalytic center of the enzyme. These studies indicate that nucleotide analogs may serve as probes for the DNA binding and catalytic sites of the enzyme and may serve as models for the design of active site inhibitors of retroviral integrase.
重组人免疫缺陷病毒1型（HIV-1）整合酶在体外被证明能结合ATP及其他核苷三磷酸和核苷酸类似物。ATP与光亲和类似物8-叠氮基-ATP和整合酶的交联以紫外线剂量依赖的方式发生。当核苷酸在与蛋白质孵育前被氧化成2'，3'-二醛衍生物（氧化ATP）时，即使没有紫外线照射，ATP与整合酶的共价结合也能实现，这表明在该蛋白质的核苷酸结合区域存在一个具有反应活性的赖氨酸残基。大量实验观察表明，核苷酸和DNA底物在整合酶蛋白的相同或重叠位点结合。例如，核苷酸或核苷酸类似物与整合酶的结合会被预先与DNA底物孵育所阻断，并且8-叠氮基-ATP与整合酶的共价交联会抑制该蛋白质的DNA结合和寡核苷酸切割活性。氧化ATP在对DNA结合无影响的浓度下抑制整合酶的寡核苷酸切割活性，这表明氧化核苷酸可能特异性靶向该酶的催化中心。这些研究表明，核苷酸类似物可作为该酶DNA结合和催化位点的探针，并可作为设计逆转录病毒整合酶活性位点抑制剂的模型。
