Spectroscopic studies on cobalt(II)-substituted metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria
Spectroscopic studies on cobalt(II)-substituted metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria
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DOI：
10.1021/bi047463s
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发表时间：
2005-04-05
期刊：
影响因子：
2.9
通讯作者：
Crowder, MW
中科院分区：
文献类型：
--
作者：
Crawford, PA;Yang, KW;Crowder, MW
为了探究 Bb 族金属-β-内酰胺酶的结构，制备了 Co(II) 取代的 ImiS，并通过电子吸收、NMR 和 EPR 光谱进行了表征。含有 1 当量 Co(II) 的 ImiS (Co(II)(1)-ImiS) 具有催化活性。 Co(II)(1)-ImiS 的电子吸收研究揭示了两个不同特征的存在：(1) 强烈的硫到 Co(II) 配体到金属电荷转移带和 (2) 强度较低的 Co(II) 配体场跃迁，表明 Co(II)(1)-ImiS 中存在 4 配位 Co(II)。 Co(II)I-ImiS 的 H-1 NMR 研究表明，一种组氨酸、一种天冬氨酸和一种半胱氨酸与 Co(II)I-ImiS 中的金属离子配位。将第二个 Co(II) 添加到 Co(II)I-ImiS 中不会导致任何额外的溶剂可交换 NMR 共振，这强烈表明第二个 Co(II) 不与具有组氨酸配体的位点结合。 EPR 研究表明，Co(II)(1)-ImiS 中的金属离子为 4 配位，第二个 Co(II) 为 5/6 配位。总而言之，这些数据表明 ImiS 中的催化位点是共有的 Zn-2 位点，其中 Co(II)（以及通过外推的 Zn(II)）为 4 配位，并与 Cys221、His263、Asp120 和可能的一个溶剂水分子结合。这些研究还表明，第二种抑制性金属离子不与共有 Zn-1 位点结合，并且金属离子结合在明显远离活性位点的位点。这些结果给出了金属-β-内酰胺酶 ImiS 的第一个结构信息，并表明 ImiS 中的第二个金属结合位点可能是抑制剂的目标。
In an effort to probe the structure of a group Bb metallo-beta-lactamase, Co(II)-substituted ImiS was prepared and characterized by electronic absorption, NMR, and EPR spectroscopies. ImiS containing 1 equiv of Co(II) (Co(II)(1)-ImiS) was shown to be catalytically active. Electronic absorption studies of Co(II)(1)-ImiS revealed the presence of two distinct features: (1) an intense sulfur to Co(II) ligand to metal charge transfer band and (2) less intense, Co(II) ligand field transitions that suggest 4-coordinate Co(II) in Co(II)(1)-ImiS. H-1 NMR studies of Co(II)I-ImiS suggest that one histidine, one aspartic acid, and one cysteine coordinate the metal ion in Co(II)I-ImiS. The addition of a second Co(II)to Co(II)I-ImiS did not result in any additional solvent-exchangeable NMR resonances, strongly suggesting that the second Co(II) does not bind to a site with histidine ligands. EPR studies reveal that the metal ion in Co(II)(1)-ImiS is 4-coordinate and that the second Co(II) is 5/6 coordinate. Taken together, these data indicate that the catalytic site in ImiS is the consensus Zn-2 Site, in which Co(II) (and by extrapolation Zn(II)) is 4-coordinate and bound by Cys221, His263, Asp120, and probably one solvent water molecule. These studies also show that the second, inhibitory metal ion does not bind to the consensus Zn-1 site and that the metal ion binds at a site significantly removed from the active site. These results give the first structural information on metallo-beta-lactamase ImiS and suggest that the second metal binding site in ImiS may be targeted for inhibitors.