Characterization of the metal-binding sites of the beta-lactamase from Bacteroides fragilis
Characterization of the metal-binding sites of the beta-lactamase from Bacteroides fragilis
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DOI：
10.1021/bi960976h
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发表时间：
1996-09-17
期刊：
影响因子：
2.9
通讯作者：
Benkovic, SJ
中科院分区：
文献类型：
--
作者：
Crowder, MW;Wang, ZG;Benkovic, SJ
为了更好地了解脆弱拟杆菌中金属- β -内酰胺酶的结构和功能，对该酶的天然形式、金属取代形式和突变形式进行了光谱和金属结合研究。原子吸收研究表明，原生B. fragilis酶与2 mol Zn（II）紧密结合，并且与诱变研究一起表明，两种金属离子的存在是充分催化活性所必需的。EPR光谱证实，Co（II）取代β -内酰胺酶每摩尔酶结合2 mol Co(II)，两个Co（II）为高自旋且可能不偶联，表观g值分别为6.5、4.2和2.01,Co（II）的配位数为5或6。该Co（II）取代酶的配体数量由紫外可见光谱证实，该光谱显示在550和650 nm之间存在非常弱的d-d跃迁（epsilon约为30 M(-1)）。cm(-1))和320 am的强烈特征（epsilon接近1570 M(-1)）。厘米(1))。后者被分配到一个半胱氨酸硫到Co（II）配体到金属的电荷转移带上，这一分配通过该带在Co（II）取代的C168S突变体的紫外-可见光谱中消失而得到证实。对Co（II）取代酶的H-1核磁共振研究表明存在三个与Co（II）结合的组氨酸配体。综上所述，这些研究支持Rasmussen等人的序列比较研究，其中存在一个具有三个组氨酸和一个半胱氨酸的催化金属结合位点（C168）。其余的配体假定是参与催化作用的水分子。诱变研究，结合活性测定和金属结合研究，已经确定Asp61、Asp90、Asp152和Asp183可能是第二个金属结合位点的配体，其中Asp90和Asp152对k（cat）有明显的影响。根据蜡样芽孢杆菌金属-内酰胺酶的最新晶体结构对这些结果进行了讨论。
In an effort to better understand the structure and function of the metallo-beta-lactamase from Bacteroides fragilis, spectroscopic and metal-binding studies were performed on the native, metal-substituted, and mutant forms of the enzyme. Atomic absorption studies demonstrate that the native B. fragilis enzyme tightly binds 2 mol of Zn(II) and, along with mutagenesis studies, that the presence of both metal ions is required for full catalytic activity. EPR spectroscopy was used to confirm that the Co(II)-substituted beta-lactamase binds 2 mol of Co(II) per mole of enzyme, that the two Co(II)'s are high-spin and probably uncoupled, with apparent g values of 6.5, 4.2, and 2.01 and that the coordination number of the Co(II) is 5 or 6. This number of ligands for the Co(II)-substituted enzyme is confirmed by UV-Vis spectra, which demonstrate the presence of very weak d-d transitions between 550 and 650 nm (epsilon approximate to 30 M(-1). cm(-1)) and an intense feature at 320 am (epsilon approximate to 1570 M(-1). cm(-1)). The latter is assigned to a cysteine sulfur to Co(II) ligand-to-metal charge transfer band, and this assignment is confirmed by the disappearance of this band in the UV-Vis spectrum of a Co(II)-substituted C168S mutant. H-1 NMR studies on the Co(II)-substituted enzyme suggest the presence of three histidine ligands bound to Co(II). Taken together, these studies support the sequence comparison study of Rasmussen et al., in which there is a catalytic metal-binding site with three histidines and one cysteine (C168). The remaining ligands are postulated to be water molecules involved in catalysis. Mutagenesis studies, in combination with activity assays and metal-binding studies, have been used to identify Asp61, Asp90, Asp152, and Asp183 as possible ligands to the second metal-binding site, with Asp90 and Asp152 having a pronounced effect on k(cat). These results are discussed in light of the recent crystal structure of the metallo-beta-lactamase from B. cereus.