Probing the Role of Asp-120(81) of Metallo-β-lactamase (IMP-1) by Site-directed Mutagenesis, Kinetic Studies, and X-ray Crystallography*
Probing the Role of Asp-120(81) of Metallo-β-lactamase (IMP-1) by Site-directed Mutagenesis, Kinetic Studies, and X-ray Crystallography*
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DOI：
10.1074/jbc.m414314200
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发表时间：
2005-05
影响因子：
4.8
通讯作者：
Y. Yamaguchi;Takahiro Kuroki;Hisami Yasuzawa;Toshihiro Higashi;Wanchun Jin;Akiko Kawanami;Y. Yamagata;Y. Arakawa;M. Goto;H. Kurosaki
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文献类型：
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作者：
Y. Yamaguchi;Takahiro Kuroki;Hisami Yasuzawa;Toshihiro Higashi;Wanchun Jin;Akiko Kawanami;Y. Yamagata;Y. Arakawa;M. Goto;H. Kurosaki
Metallo-β-lactamase IMP-1 是一种二锌 (II) 金属酶，可有效水解 β-内酰胺抗生素。野生型(WT) IMP-1的活性位点具有保守的Asp-120(81)，在催化中发挥重要作用。为了探讨Asp-120(81)对IMP-1的催化作用，通过定点诱变制备了IMP-1突变体D120(81)A和D120(81)E，并进行了各种动力学研究。尽管 IMP-1 突变体的活性位点含有两个 Zn(II) 离子，但与 WT 相比，IMP-1 突变体的 kcat 值下降了 102-104 倍。为了评估 Asp-120(81) 的酸碱特性，通过停流研究检查了水解的 pH 依赖性。对于 WT 和 D120(81)A，在 pH 5 和 9 之间没有发现可观察到的 pKa 值。等摩尔量的硝基头孢菌素和所有酶的快速混合未能导致在 650 nm 处检测到硝基头孢菌素的阴离子中间体。这些结果表明，IMP-1 的 Asp-120(81) 不是降低桥接两个 Zn(II) 离子的水的 pKa 的因素，也不是阴离子中间体的质子供体。就 D120(81)E 而言，头孢硝酯水解产物在 460 nm 处显示最大吸收，以质子化形式与 D120(81)E 结合。 D120(81)A 和 D120(81)E 的三维结构也分别以 2.0 和 3.0 Å 的分辨率测定。在D120(81)E的情况下，由于Glu-120(81)OE1配位模式的变化以及活性位点保守的His-263(197)的位置移动，Zn-Zn距离比WT增加了0.3 Å。
Metallo-β-lactamase IMP-1 is a di-Zn(II) metalloenzyme that efficiently hydrolyzes β-lactam antibiotics. Wild-type (WT) IMP-1 has a conserved Asp-120(81) in the active site, which plays an important role in catalysis. To probe the catalytic role of Asp-120(81) in IMP-1, the IMP-1 mutants, D120(81)A and D120(81)E, were prepared by site-directed mutagenesis, and various kinetics studies were conducted. The IMP-1 mutants exhibited 102–104-fold drops in kcat values compared with WT despite the fact that they contained two Zn(II) ions in the active site. To evaluate the acid-base characteristics of Asp-120(81), the pH dependence for hydrolysis was examined by stopped-flow studies. No observable pKa values between pH 5 and 9 were found for WT and D120(81)A. The rapid mixing of equimolar amounts of nitrocefin and all enzymes failed to result in the detection of an anion intermediate of nitrocefin at 650 nm. These results suggest that Asp-120(81) of IMP-1 is not a factor in decreasing the pKa for the water bridging two Zn(II) ions and is not a proton donor to the anionic intermediate. In the case of D120(81)E, the nitrocefin hydrolysis product, which shows a maximum absorption at 460 nm, was bound to D120(81)E in the protonated form. The three-dimensional structures of D120(81)A and D120(81)E were also determined at 2.0 and 3.0 Å resolutions, respectively. In the case of D120(81)E, the Zn-Zn distance was increased by 0.3 Å compared with WT, due to the change in the coordination mode of Glu-120(81)OE1 and the positional shift in the conserved His-263(197) at the active site.