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...

When expressed by pathogenic bacteria, Zn2؉ -␤-lactamases induce resistance to most ␤-lactam antibiotics. A possible strategy to fight these bacteria would be a combined therapy with non-toxic inhibitors of Zn 2؉ -␤-lactamases together with standard antibiotics. For this purpose, it is important to verify that the inhibitor is effective under all clinical conditions. We have investigated the correlation between the number of zinc ions bound to the Zn 2؉ -␤-lactamase from Bacillus cereus and hydrolysis of benzylpenicillin and nitrocefin for the wild type and a mutant where cysteine 168 is replaced by alanine. It is shown that both the mono-Zn 2؉ (mononuclear) and di-Zn 2؉ (binuclear) Zn 2؉ -␤-lactamases are catalytically active but with different kinetic properties. The mono-Zn2؉ -␤-lactamase requires the conserved cysteine residue for hydrolysis of the ␤-lactam ring in contrast to the binuclear enzyme where the cysteine residue is not essential. Substrate affinity is not significantly affected by the mutation for the mononuclear enzyme but is decreased for the binuclear enzyme. These results were derived from kinetic studies on two wild types and the mutant enzyme with benzylpenicillin and nitrocefin as substrates. Thus, targeting drug design to modify this residue might represent an efficient strategy, the more so if it also interferes with the formation of the binuclear enzyme.

In one of the first studies of isolated intermediates in protein aggregation, we have used circular dichroism and fluorescence spectroscopy to characterize metastable oligomers that are formed in the early steps of ␤-lactoglobulin heat aggregation. The intermediates show typical molten globule characteristics (secondary structure content similar to the native and less tight packing of the side chains), in agreement with the belief that partly folded states play a key role in protein aggregation. The far-UV CD signal bears strong resemblance to that of a known folding intermediate. Cryo-transmission electron microscopy of the aggregates reveals spherical particles with a diameter of about 50 nm and an internal threadlike structure. Isolated oligomers as well as larger aggregates bind the dye thioflavin T, usually a signature of the amyloid superstructures found in many protein aggregates. This result suggests that the structural motif recognized by thioflavin T can be formed in small oligomers.

The bacterial Zn2+ metallo-beta-lactamase from B. fragilis is a zinc-enzyme with two potential metal ion binding sites. It cleaves the lactam ring of antibiotics, thus contributing to the acquired resistance of bacteria against antibiotics. The present study bears on the binuclear form of the enzyme. We compare several possible binding modes of captopril, a mercaptocarboxamide inhibitor of several zinc-metalloenzymes. Two diastereoisomers of captopril were considered, with either a D- or an L-proline residue. We have used the polarizable molecular mechanics procedure SIBFA (Sum of Interactions Between Fragments ab initio computed). Two beta-lactamase models were considered, encompassing 104 and 188 residues, respectively. The energy balances included the inter and intramolecular interaction energies as well as the contribution from solvation computed using a continuum reaction field procedure. The thiolate ion of the inhibitor is binding to both metal ions, expelling the bridging solvent molecule from the uncomplexed enzyme. Different competing binding modes of captopril were considered, either where the inhibitor binds in a monodentate mode to the zinc cations only with its thiolate ion, or in bidentate modes involving additional zinc binding by its carboxylate or ketone carbonyl groups. The additional coordination by the inhibitor's carboxylate or carbonyl group always occurs at the zinc ion, which is bound by a histidine, a cysteine, and an aspartate side chain. For both diastereomers, the energy balances favor monodentate binding of captopril via S-. The preference over bidentate binding is small. The interaction energies were recomputed in model sites restricted to captopril, the Zn2+ cations, and their coordinating end side chains from beta-lactamase (98 atoms). The interaction energies and their ranking among competing arrangements were consistent with those computed by ab initio HF and DFT procedures.

Adsorption of cationic high molecular weight polyacrylamides (CPAM) (M(w) is about 800 kDa) with different fractions of cationic units tau = 0.09 and tau = 0.018 onto silica surface was studied over a wide range of pH (4-9) and KCl concentration (c(s) = 10(-3)-10(-1) M) by in-situ null ellipsometry. We discuss how the adsorbed layer depends on the bulk conditions as well as kinetically responds to changes in solution conditions. The adsorbed amount Gamma of CPAM increases with pH for all studied electrolyte concentrations until a plateau Gamma is reached at pH > 6. At low pH we observed an increase in adsorbed amount with electrolyte concentration. At high pH there is no remarkable influence of added salt on the values of the adsorbed amount. The thickness of adsorbed polymer layers, obtained by ellipsometry, increases with electrolyte concentration and decreases with pH. At low c(s) and high pH the polyelectrolyte adsorbs in a flat conformation. An overcompensation of the surface charge (charge reversal) by the adsorbed polyelectrolyte is observed at high c(s) and low pH. To reveal the reversibility of the polyelectrolyte adsorption with respect to the adsorbed amount and layer thickness, parameters such as polyelectrolyte concentration (c(p)), c(s), and pH were changed during the experiment. Generally, similar adsorbed layer properties were obtained independent of whether adsorption was obtained directly to initially bare surface or by changing pH, c(s), or the concentration of polyelectrolyte solution in the presence of a preadsorbed layer, provided that the coverage of the preadsorbed layer was low. Once a steady state of the measured parameters (Gamma, d) was reached, experimental conditions were restored to the original values and corresponding changes in Gamma and adsorbed layer thickness were recorded. For initially low surface coverage it was impossible to restore the layer properties, and in this case we always ended up with higher coverage than the initial values. For initial high surface coverage it was usually possible to restore the initial layer properties. Thus, we concluded that polyelectrolyte appears only partially reversible to changes in the solution conditions due the slow rearrangement process within the adsorbed layer.