Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM-1

Abstract

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data.

Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity

“…Resonances at 64, 72, and 79 ppm were previously assigned to solvent-exchangeable protons on His120, His122, and His189 in the Zn1 site, while resonances at 48, 107, and 165 ppm have been assigned, respectively, to protons on Asp124, His250 and Cys208 at the Zn2 site (14). The spectrum of dicobalt (II) NDM-1 shown here ( Figure 8) reproduces what we have reported earlier (5,15).…”

“…The extinction coefficients in the low energy pair of ligand field bands are not affected significantly, while those of the higher energy pair of bands change by up to 50 %. Based on our previous work on NDM-1 (14), which showed a more symmetric set of ligand field bands with Co(II) in the Zn1 site (CoCd-NDM-1) and a more asymmetric set of bands for the Zn2 site (ZnCo-NDM-1), the data in Figure 7 may indicate that the mutations are shifting the spectra toward the Zn2 structure, which is more asymmetric and fivecoordinate. However, we believe the observation of more intense ligand fields bands is a consequence of weaker Co-S bonding, and the optical spectra are consistent with maintenance of the overall active site structure, including the ligand sets to both metal ions.…”

Kinetic, mechanistic, structural and spectroscopic investigations of Bimetallic Metallohydrolases

“…For both NDM-1 and L1 there is reasonable agreement between those theoretical values and the corresponding values measured in steady-state kinetic assays (102,128) . For AIM-1 neither model A nor Model B led to a good agreement between theoretical and experimental parameters (Table 5. (Table 5.5).…”

“…The main difference between the two models is that the steps leading to the formation and decay of the intermediate (i.e. k2 and k3, Figure 5.16) are assumed reversible in Model A and irreversible in Model B (Model A has previously been employed to analyse comparable data for the MBLs L1 and NDM-1 (102,128) (123) . The microscopic rate constants listed in Table 5.5 can be used to calculate theoretical values for kcat…”

“…Previous studies with the MBLs NDM-1 and L1 demonstrated that fluorescence measurements may be used to monitor substrate binding (102,128) . The change in fluorescence during this reaction is associated with a tryptophan residue in the vicinity of the active site, i.e.…”