Metallo-β-lactamase : Structural basis for substrate-specificity and rational design of the inhibitors

金属β-内酰胺酶（IMP-1）由于广泛的底物特异性和缺乏有效的抑制剂，对临床环境构成潜在威胁。IMP-1需要两个Zn（II）离子来水解β-内酰胺类抗生素。为了研究Zn（II）与IMP-1结合的影响，我们研究了Zn（II）与野生型IMP-1的解离动力学。从动力学实验来看，Zn（II）离子与IMP-1的解离表现为两个步骤。将EDTA直接添加到野生型IMP-1中，在30℃下制备Apo IMP-1。直到Co（II）以1:1的比例加入载脂蛋白IMP-1时，由于d-d跃迁引起的吸收带才出现。在载脂蛋白IMP-1中进一步添加Co（II）至2:1，由于Cys221的硫代盐到Co（II）的LMCT和d-d跃迁，导致344 nm处的吸收增加。这些结果表明，Co（II）与载脂蛋白IMP-1的结合是逐步进行的：Co（II）离子首先结合到Zn1位点，然后第二个Co（II）离子结合到Zn2位点。我们制备了两种抑制剂，五氟苯基3-巯基丙酸酯（PFMP, 1）和3-（3-巯基丙酸磺酰）丙酸五氟苯基酯（MPAP, 2），用于MBLs之一IMP-1。从酶抑制剂复合物的凝胶过滤实验来看，这些化合物不可逆地抑制了IMP-1。此外，x射线晶体学显示抑制剂2与IMP-1共价结合，在Lys224的氨基（N^ζ）与抑制剂之间形成酰胺键。

Metallo-β-lactamase (IMP-1) poses a potential threat for clinical environment because of wide spread substrate specificity and lack of the available inhibitors. IMP-1 requires two Zn(II) ions to hydrolyze β-lactam antibiotics.To study the effects on Zn(II) binding to IMP-1, we examined the kinetics of dissociation of Zn(II) from wild type IMP-1. From the kinetic experiments, the dissociation of Zn(II) ion from IMP-1 appeared to be two steps. Apo IMP-1 was prepared by the addition of EDTA directly to wild type IMP-1 at 30 ℃. Up to the addition of Co(II) to apo IMP-1 in 1:1, the absorption bands due to d-d transitions appeared. Further addition of Co(II) to apo IMP-1 up to 2:1 resulted in the increases in absorption at 344 nm, due to LMCT from thiolate of Cys221 to Co(II), and d-d transitions. These result suggest that the binding of Co(II) to apo IMP-1 proceeds stepwise : a Co(II) ion initially binds to the Zn1 site and then the second Co(II) ion binds to the Zn2 site.We prepared two inhibitors, pentafluorophenyl 3-mercaptopropionate (PFMP, 1) and 3-(3-mercaptopropionylsulfanyl)propionic acid pentafluorophenyl ester (MPAP, 2) for one of MBLs, IMP-1. From the gel-filtration experiment of the enzyme-inhibitor complex, these compounds inhibited IMP-1 irreversibly. Moreover, X-ray crystallography revealed that inhibitor 2 covalently binds to IMP-1 to form an amide bond between the amino group (N^ζ) of Lys224 and the inhibitor.