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

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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.