Bujacz G, Alexandratos J, Wlodawer A, Merkel G, Andrake M, Katz R A, Skalka A M
Macromolecular Structure Laboratory, NCI-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Frederick, Maryland 21702, USA.
J Biol Chem. 1997 Jul 18;272(29):18161-8. doi: 10.1074/jbc.272.29.18161.
Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called "processing," and the second is a polynucleotide transfer or "joining" step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences.
逆转录病毒整合酶（INs）包含两个已知的金属结合结构域。N端结构域包含一个锌指基序，已证明其能结合Zn2+，而中央催化核心结构域包含一个酸性氨基酸三联体，可结合Mn2+或Mg2+，这两种金属是酶活性所需的金属辅因子。整合反应分两个不同步骤进行；第一步是一个特定的核酸内切酶切割步骤，称为“加工”，第二步是多核苷酸转移或“连接”步骤。我们之前的结果表明，禽肉瘤病毒IN体外活性的金属偏好为Mn2+>Mg2+，并且在分离的催化结构域晶体中，三种关键活性位点残基（Asp-64和Asp-121）中的两个可与单一金属阳离子配位。在此，我们报告Ca2+、Zn2+和Cd2+也可结合在催化结构域的活性位点。此外，两个锌和镉阳离子结合在活性位点，活性位点三联体的所有三个残基（Asp-64、Asp-121和Glu-157）都参与它们的配位。这些结果与逆转录病毒整合酶催化的双金属机制一致。我们还表明，Zn2+可作为禽肉瘤病毒IN全长蛋白、缺乏N端结构域的衍生物或分离的催化结构域催化的核酸内切酶反应的辅因子。然而，在存在Zn2+的情况下，多核苷酸转移酶活性严重受损或无法检测到。因此，尽管整合酶的加工和连接步骤采用相似机制和相同的活性位点三联体，但它们可通过金属偏好明显区分。
