One high affinity (nM) and one low affinity (M) macroscopic dissociation constant for the binding of metal ions were found for the wild-type metallo-␤-lactamase from Bacillus cereus as well as six single-site mutants in which all ligands in the two metal binding sites were altered. Surprisingly, the mutations did not cause a specific alteration of the affinity of metal ions for the sole modified binding site as determined by extended x-ray absorption fine structure (EXAFS) and perturbed angular correlation of ␥-rays spectroscopy, respectively. Also UV-visible absorption spectra for the mono-cobalt enzymes clearly contain contributions from both metal sites. The observations of the very similar microscopic dissociation constants of both binding sites in contrast to the significantly differing macroscopic dissociation constants inevitably led to the conclusion that binding to the two metal sites exhibits negative cooperativity. The slow association rates for forming the binuclear enzyme determined by stopped-flow fluorescence measurements suggested that fast metal exchange between the two sites for the mononuclear enzyme hinders the binding of a second metal ion. EXAFS spectroscopy of the mono-and di-zinc wild type enzymes and two di-zinc mutants provide a definition of the metal ion environments, which is compared with the available x-ray crystallographic data.Two zinc binding sites in close proximity are conserved in all metallo-␤-lactamases studied so far. Only two of the metal ion ligands undergo variations between the three different subclasses of the enzyme family (1). The enzyme from Bacillus cereus 569/H/9 (BcII) 1 represents a member of subclass B1 with 3 His ligands in one site and 1 Asp, 1 Cys, and 1 His ligand in the other site (3H 1 and DCH 1 sites, respectively). Various crystal structures of BcII are available, representing mononuclear (2) and binuclear species (3, 4). It was shown earlier that both mono-and binuclear zinc enzymes from B. cereus (5) and Bacteroides fragilis (6) are catalytically active.Although catalytic mechanisms for the enzyme with either one or two zinc ions bound have been discussed (for review see Ref. 7) the respective roles of the two binding sites during catalysis are still unclear. Generally the 3H site is considered to be the primary catalytic site. However, the importance of the DCH site for catalysis became obvious from studies of the C168A mutant. When only one zinc ion is bound to this mutant, it shows a very low activity compared with the wild type, whereas wild type-like activity is almost restored when a second metal ion is bound (5).Perturbed angular correlation (PAC) of ␥-ray spectroscopy provides information on the metal ion coordination geometry through measurement of the nuclear quadrupole interaction (NQI) between the nuclear electric quadrupole moment and the electric field gradient from the surrounding charge distribution. With this method it was possible to demonstrate that the Cd(II) ions in the mononuclear wild type BcII are distributed between the two m...

D-and L-captopril are competitive inhibitors of metallo-␤-lactamases. For the enzymes from Bacillus cereus (BcII) and Aeromonas hydrophila (CphA), we found that the mononuclear enzymes are the favored targets for inhibition. By combining results from extended x-ray absorption fine structure, perturbed angular correlation of ␥-rays spectroscopy, and a study of metal ion binding, we derived that for Cd(II) 1 -BcII, the thiolate sulfur of D-captopril binds to the metal ion located at the site defined by three histidine ligand residues. This is also the case for the inhibited Co(II) 1 and Co(II) 2 enzymes as observed by UV-visible spectroscopy. Although the single metal ion in Cd(II) 1 -BcII is distributed between both available binding sites in both the uninhibited and the inhibited enzyme, Cd(II) 1 -CphA shows only one defined ligand geometry with the thiolate sulfur coordinating to the metal ion in the site composed of 1 Cys, 1 His, and 1 Asp. CphA shows a strong preference for D-captopril, which is also reflected in a very rigid structure of the complex as determined by perturbed angular correlation spectroscopy. For BcII and CphA, which are representatives of the metallo-␤-lactamase subclasses B1 and B2, we find two different inhibitor binding modes.Metallo-␤-lactamases confer antibiotic resistance to bacteria by catalyzing the hydrolysis of ␤-lactam antibiotics, including carbapenems. This relatively new form of resistance is spreading and thereby escaping the effective inhibitors developed to fight the better known serine-␤-lactamases. For all metallo-␤-lactamases investigated, structurally similar enzyme active sites comprising two zinc binding sites are reported. For Bacillus cereus metallo-␤-lactamase (BcII), 1 one metal-binding site contains three His (H-site); the other one contains 1 Asp, 1 Cys, and 1 His as the metal ligating residues (DCH site) as derived from x-ray crystallography (1). For CphA, 1 His from the H-site (His-116) is supposed to be replaced by an Asn (2). Various thiol-carboxylate compounds were identified as potent inhibitors (3). The active site binding of thiomandelic acid to BcII was studied by NMR spectroscopy (4), whereas the binding of 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mercaptomethyl)-4-(phenylbutrylglycine)] to the enzyme from Pseudomonas aeruginosa (IMP-1) was characterized by x-ray crystallography (5). With both approaches, the inhibited binuclear zinc enzymes were studied. Both studies agree in a bridging role of the metal-bound sulfur of the inhibitor, whereas the carboxylate group of the inhibitors binds to an accessible amino acid, thus stabilizing the complex. Other known inhibitory compounds are 2,3-(S,S)-disubstituted succinic acids for IMP-1 (6) or moxalactam and cefoxitin for CphA (7). The latter compounds lead to irreversible inactivation of the enzyme by the hydrolyzed reaction products.The structural investigation of D-and L-captopril binding presented here is based on results obtained from enzyme kinetic and thermodynamic studies. Captopril is known as an ...

The two metal sites in cadmium substituted beta-lactamase from Bacillus cereus 569/H/9 have been studied by NMR spectroscopy ((1)H, (15)N, and (113)Cd) and PAC spectroscopy ((111m)Cd). Distinct NMR signals from the backbone amides are identified for the apoenzyme and the mononuclear and binuclear cadmium enzymes. For the binuclear cadmium enzyme, two (113)Cd NMR signals (142 and 262 ppm) and two (111m)Cd PAC nuclear quadrupole interactions are observed. Two nuclear quadrupole interactions are also observed, with approximately equal occupancy, in the PAC spectra at cadmium/enzyme ratios < 1; these are different from those derived for the binuclear cadmium enzyme, demonstrating interaction between the two metal ion binding sites. In contrast to the observation from PAC spectroscopy, only one (113)Cd NMR signal (176 ppm) is observed at cadmium/enzyme ratios < 1. The titration of the metal site imidazole (N)H proton signals as a function of cadmium ion-to-enzyme ratio shows that signals characteristic for the binuclear cadmium enzyme appear when the cadmium ion-to-enzyme ratio is between 1 and 2, whereas no signals are observed at stoichiometries less than 1. The simplest explanation consistent with all data is that, at cadmium/enzyme ratios < 1, the single Cd(II) is undergoing exchange between the two metal sites on the enzyme. This exchange must be fast on the (113)Cd NMR time scale and slow on the (111m)Cd PAC time scale and must thus occur in a time regime between 0.1 and 10 micros.

We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-␤-lactamase subclasses, B1 (BcII from Bacillus cereus and BlaB from Chryseobacterium meningosepticum), B2 (CphA from Aeromonas hydrophila), and B3 (L1 from Stenotrophomonas maltophilia). By competition experiments with metal-free apoenzymes and chromophoric zinc chelators or EDTA, we determined the dissociation constants in the absence and presence of substrates. For the formation of the monozinc enzymes we determined constants of 1.8, 5.1, 0.007, and 2.6 nM in the absence and 13.6, 1.8, 1.2, and 5.7 pM in the presence of substrates for BcII, BlaB, CphA, and L1, respectively. A second zinc ion binds in the absence (presence) of substrates with considerably higher dissociation constants, namely 1.8 (0.8), 0.007 (0.025), 50 (1.9), and 0.006 (0.12) M for BcII, BlaB, CphA, and L1, respectively. We have concluded that the apo form might be the prevailing state of most of the metallo-␤-lactamases under physiological conditions in the absence of substrates. Substrate availability induces a spontaneous self-activation due to a drastic decrease of the dissociation constants, resulting in the formation of active mononuclear enzymes already at picomolar free zinc ion concentrations. In the presence of substrates, the binuclear state of the enzymes only exists at unphysiologic high zinc concentrations and might be of no biological relevance. From the competition experiments with EDTA it is further concluded that the reactivation rate does not depend on the pool of free zinc ions but proceeds via the EDTA-Zn(II)-enzyme ternary complexes.Metallo-␤-lactamases (class B ␤-lactamases) are produced by bacteria as extracellular or periplasmatic enzymes. All known representatives possess two conserved metal binding sites and require zinc ions as enzymatic cofactors. By catalyzing the hydrolysis of ␤-lactams they render the corresponding strains resistant to almost all ␤-lactam antibiotics. Their increasing emergence in pathogenic bacterial strains due to a rapid dissemination by horizontal gene transfer has induced a growing interest in this enzyme family because of the lack of efficient therapies to treat infected patients.The metallo-␤-lactamases constitute a group of heterogeneous proteins that is divided into subclasses B1, B2, and B3 (1). Subclass B1 exhibits a broad substrate profile (2), and its zinc binding sites are composed of His-116, His-118, and His-196 (site 1) and Asp-120, Cys-221, and His-263 (site 2) (numbering according to Ref. 3). In subclass B2 the zinc ligands in site 2 are conserved, whereas His-116 in site 1 is replaced by Asn. Representatives of subclass B2 efficiently hydrolyze only carbapenems (2) and are active as monozinc enzymes, whereas the binding of a second zinc ion causes non-competitive inhibition (4). In subclass B3, Cys-221 is substituted by a Ser and is replaced by His-121 as a zinc ligand in site 2. In the Gob-1 enzyme (Chryseobacterium meningosepticum PINT) an additional H...