Rogert Bauer

MONO- AND BINUCLEAR ZN2+-BETA -LACTAMASE : ROLE OF THE CONSERVED CYSTEINE IN THE CATALYTIC MECHANISM

When expressed by pathogenic bacteria, Zn2+-β-lactamases induce resistance to most β-lactam antibiotics. A possible strategy to fight these bacteria would be a combined therapy with non-toxic inhibitors of Zn2+-β-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 Zn2+-β-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-Zn2+ (mononuclear) and di-Zn2+(binuclear) Zn2+-β-lactamases are catalytically active but with different kinetic properties. The mono-Zn2+-β-lactamase requires the conserved cysteine residue for hydrolysis of the β-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.

Mono- and binuclear Zn-β-lactamase from Bacteroides fragilis: catalytic and structural roles of the zinc ions

The Bacteroides fragilis Zn‐β‐lactamase is active with a mono‐ and a binuclear zinc site. The apoenzyme produced by removal of both Zn ions does not recover full activity upon readdition of Zn2+ in contrast to an active mono‐Zn form prepared at pH 6.0. Differences in k cat values observed are substrate‐dependent implying distinct mechanisms for the mono‐ and binuclear species. The substrate profile of a Zn,Cd hybrid obtained by selective exchange of one zinc ion is different from that of the Zn2 enzyme with a remarkable 15‐fold increased activity with cefoxitin as substrate.

Analysis of perturbed angular correlation spectra of metal ions bound to proteins with rotational correlation times in the intermediate region

The formulation by Dattagupta of the strong-collision model, describing the effect on the perturbation function,G2(t) by the isotropic tumbling of an electric field gradient, is generalized to electric field gradients with no axial symmetry. The effect on the perturbation function by strong collisions is compared to the effect of rotational diffusion in the adiabatic limit. The comparison is carried out for decays with an intermediate state of spin 5/2 and for non-axially symmetric electric field gradients. It shows that the strong-collision model can be used for interpretation of PAC spectra of molecules with correlation times between the adiabatic and the fast relaxation limits. The strongcollision model is then used to determine the rotational diffusion of the cadmium substituted copper, zinc superoxide dismutase at 3°C and 25°C from111mCd TDPAC spectra. For these analyses, the model is incorporated into a conventional least-squares fitting routine.

Structural information concerning the catalytic metal site in horse liver alcohol dehydrogenase, obtained by perturbed angular correlation spectroscopy on 111Cd

Perturbed angular correlation spectra on horse liver alcohol dehydrogenase, measured on 111Cd inserted specifically in the catalytic site, have been obtained under various conditions. Spectra have been obtained in the pH range 6–9 and in the presence of coenzyme and/or pyrazole or trifluoroethanol No ionization at the metal site could be detected between pH 6 and 9, irrespectively of whether NAD+ is present or not. From this we conclude that a water molecule ligated to Cd(II) must have a pK of ionization higher than 9.5. Both coenzyme, pyrazole and trifluoroethanol affect the spectra of Cd incorporated in the catalytic site of alcohol dehydrogenase. The interpretation of the present data is consistent with a four coordinated, nearly tetrahedral metal geometry, both with and without the coenzyme bound to the enzyme. The effect of the coenzyme on the metal coordination can best be explained by a reduction of the two cysteine metal bond lengths. From an analysis of the data we conclude that both pyrazole and trifluoroethanol enter as a fifth ligand not displacing the solvent ligand to the metal.

The potential of perturbed angular correlation of gamma rays as a tool for dynamic studies of peptides/proteins

Four peptides and serum albumin have been derivatized with the bicyclic anydride of diethylenetriamine-pentaacetic acid. Procedures were developed to isolate the labelled species and determine the degree of derivatization. By using perturbed angular correlation of gamma-ray spectroscopy it is possible, through the determination of rotational correlation times, to decide whether labelled peptides interact with other molecules (receptors). In the case of the peptide cholecystokinin it is shown that the interaction between the peptide and its corresponding polyclonal antibody can be determined down to 1 pmol hormone. Experiments on 111In- and 111mCd-labelled Gly-Trp showed that, where the 111Cd PAC spectrum directly reflected the rotational motion of the molecule, the 111In PAC spectrum was affected by the nuclear transitions to 111mCd.

Nuclear quadrupole interactions in cadmium complexes : Semiempirical and ab initio calculations

Abstract Semiempirical calculations, based on the so-called angular overlap model, have been compared with ab initio methods (MP2) for the calculation of nuclear quadrupole interactions (NQI’s) in cadmium complexes with biologically relevant ligands (H2O, OH-, cysteinate, carboxylate, and imidazole). The assumptions on which the semiempirical model is based have been tested and the comparison indicates that: 1) A change in the Cd-ligand bond length by 0.1 A may change the electric field gradient (EFG) by about 0.2 a. u.. A simple scheme to incorporate such effects in the semiempirical method is suggested. 2) The effect of ligand-ligand interactions is up to about 0.2 a. u. for the largest diagonal element of the EFG tensor for the tested complexes, and such effects can significantly influence the so-called asymmetry parameter. 3) The position of non-coordinating atoms on the ligands can in some cases (e. g. the hydrogen atoms of water) significantly influence the EFG. The combined effect of non-coordinating atoms and ligand-ligand interactions may cause deviations of up to 0.35 a.u. between ab initio and the semiempirical calculations. 4) In the semiempirical model each ligand is characterised by one parameter, the so-called partial nuclear quadrupole interaction. This parameter has been evaluated by ab initio calculations, and agreement was found within about 0.2 a. u. (≈ 40 Mrad/s) for all ligands except imidazole. 5) A change in the coordination number from 2 to 6 may change the partial NQI by about 0.3 a. u.

Nuclear quadrupole interaction of111Cd on type-1 Cu-sites in blue copper proteins

The nuclear quadrupole interaction (NQI) of111Cd substituted for Cu(II) on type-1 sites in blue copper proteins is characterized by high values of ω0 in the region of 300 Mrad/s, close to that for the catalytic zinc site in alcohol dehydrogenase. Type-1 Cu has usually two sulfur ligands and two nitrogen ligands and in some cases an oxygen ligand in either a distorted tetrahedral geometry or in a trigonal bipyramidal geometry. The near tetrahedral arrangement together with the ligand sphere containing the same number of sulfur ligands explains the value of ω0 in the blue copper proteins. The present work determined the partial NQI for methionine using the known structure of azurin. This value was then used in the angular overlap model to calculate the NQI for ascorbate oxidase the structure of which is also known and gave good agreement with experiment. NQI data for laccase and stellacyanin the structures of which are unknown, are also given.

Small Scale Intramolecular Flexibility in 111mCd-Plastocyanin

Abstract The effect of mutations in the vicinity of the putative electron transfer path on the metal center of the electron transfer protein plastocyanin (spinacea) is investigated by monitoring the nuclear quadrupole interaction of 111mCd in Cd-derivatives of the protein via time differential perturbed angular correlation. The spectra for the wild type protein and the mutants were rather similar. All spectra exhibit a peculiar line profile which points towards a small scale intramolecular flexibility of the metal center.

Reduced rate of uptake of zinc ions in a calf affected with the lethal syndrome A46 relative to clinically normal calves using whole body radio-isotope scanning of 62Zn

Four clinically healthy control calves were scanned and blood clearance was determined from blood samples taken during the first hour after intravenous injection of 62Zn. Furthermore, one clinically healthy control calf and one calf suffering from the lethal hereditary syndrome A46 were scanned and blood samples taken from five min after intravenous and abomasal injection of 62Zn and onwards for a period of up to 26th. The results show a very fast liver uptake of 62Zn. The estimated rate of plasma/blood clearance and liver uptake for the control calves agrees with values derived from studies on other animals. In contrast, the A46 calf had a decreased rate of plasma/blood clearance and liver uptake relative to the control calves.