Molecular dynamics simulations of the dinuclear zinc-beta-lactamase from Bacteroides fragilis complexed with imipenem

Herein, we present results from MD simulations of the Michaelis complex formed between the dizinc beta-lactamase from B. fragilis and imipenem. We considered two catalytically important configurations, which differ in the presence or absence of a hydroxide bridge connecting the two zinc ions in the active site. The structural and dynamical effects induced by substrate binding, the specific roles of the conserved residues and the zinc-bound water molecules, the near attack conformers of the Michaelis complex, and so forth, are discussed in detail. The relative stability of the two configurations was estimated from QM linear scaling calculations on the enzyme-substrate complex combined with Poisson-Boltzmann electrostatic calculations and normal mode calculations. Importantly, we find that the two configurations have similar energies, indicating that these two structures could readily be interchanged, thereby facilitating catalysis. The configuration with the hydroxide bound to the two zinc ions is predicted to be the resting form of the enzyme, while the configuration without the bridge is the reactive form that was found to place the hydroxide in position to attack the carbonyl of the beta-lactam ring. Thus, we propose that the enzyme initiates catalysis by converting from the hydroxide bridge form into the configuration that lacks the hydroxide bridge. This interconversion increases the nucleophilicity of the hydroxide ion and exposes it to the beta-lactam carbonyl, which ultimately facilitates nucleophilic attack. The implications of the observed modes of binding, the possible influence of mutating the Lys184 and Asn193 residues on substrate binding, and the reaction mechanism are also discussed in detail.     

Sensitive determination of diquat by a kinetic method using the stopped-flow mixing technique

A simple, sensitive, selective, fast and inexpensive assay for the determination of diquat is proposed. The method is based on the reduction of the herbicide to a strongly fluorescent monocation radical with sodium dithionite. The initial rate of this reaction is directly proportional to the diquat concentration. The stopped-flow mixing technique was used because the kinetic data can be obtained in only 7 s, meaning that the method can be automated. The calibration graph is linear over the range 5-500 ng ml-1 and the precision (RSD) is close to 1.2%. The applicability of the method was demonstrated by determining the herbicide in different kinds of samples.     

Potentiometric study of the protonation and distribution equilibria of d-gluconic-delta-lactone acid in sodium perchlorate solutions at 25 degrees C and construction of a thermodynamic model

The protolytic behavior of d-gluconic-delta-lactone acid has been studied by means of automated potentiometric titrations at different ionic strengths in the range 0.1相似文献   

Role of the tautomerism of 2-azaadenine and 2-azahypoxanthine in substrate recognition by xanthine oxidase

The tautomerism of 2-azaadenine and 2-hypoxanthine has been examined in the gas phase and in aqueous solution. The tautomerism in the gas phase has been studied by means of semiempirical and ab initio quantum-mechanical computations, as well as density-functional calculations. The influence of the aqueous solvent on the relative stability between tautomers has been estimated from self-consistent reaction field calculations performed with different high-level continuum models. The results provide a detailed picture of the tautomeric preference for these purine bases. The importance of tautomerism in the substrate recognition by xanthine oxidase is discussed. Finally, the rate of oxidation of 2-azaadenine and 2- hypoxanthine by xanthine oxidase is discussed in terms of the recognition model at the enzyme active site.     

Flow injection analysis: Rayleigh light scattering technique for total protein determination

A novel flow injection analysis (FIA) method with Rayleigh light scattering (RLS) detection was developed for the determination of total protein concentrations. This method is based on the weak intensity of RLS of bromothymol blue (BB) (3',3"-dibromothymolsulfonephthalein) which can be enhanced by the addition of protein in weakly acidic solution. A common spectrofluorimeter was used as a detector. It was proved that the application of this method to quantify the total proteins in real samples by using bovine serum albumin was possible. The RLS signal was detected at lambda(ex)= lambda(em)=572 nm. The linear range was 7.0-70.0 microg mL(-1), the detection limit was 3.75 microg mL(-1), the reproducibility was 5.5% (n=7), and the sample throughput was 26 h(-1).     

Thermal behaviour of advanced composite materials based on SiC fibres

Emanation thermal analysis (ETA) was used for characterization of thermal behaviour of SiC_f/SiC composites on heating in argon and air, respectively. Effect of gas environment (argon, air) and helium ions implantation on the microstructure development of the SiC_f/SiC composite prepared by chemical vapour infiltration (CVI) from Nicalon CG fibres was investigated under in situ conditions of heating. The annealing of near surface structure irregularities was observed in the range 280-700°C and evaluated by means of the mathematical model, assuming that the structure irregularities served as diffusion paths for radon. The ETA reflected the formation of amorphous silica and its subsequent crystallization to crystoballite. Morphology of the SiC_f/SiC samples before and after the heat treatments was characterized by means of SEM. This revised version was published online in July 2006 with corrections to the Cover Date.     

Complexity of intercalation of hydrazine into kaolinite--a controlled rate thermal analysis and DRIFT spectroscopic study

Controlled rate thermal analysis (CRTA) allows the separation of adsorbed and intercalated hydrazine. CRTA displays the presence of three different types of hydrogen-bonded hydrazine in the intercalation complex: (a) The first is adsorbed loosely bonded on the kaolinite structure fully expanded by hydrazine-hydrate and liberated between approx 50 and 70 degrees C (b) The second intercalated hydrazine is lost between approx 70 and 85 degrees C. (c) The third type of intercalated-hydrazine molecule is lost in the 85-130 degrees C range. CRTA at 70 degrees C enables the removal of hydrazine-water and results in the partial collapse of the hydrazine-intercalated kaolinite structure to form a hydrazine-intercalated kaolinite. Removal of the adsorbed hydrazine enables the DRIFT spectra of the hydrazine-intercalated complex without any adsorbed hydrazine to be obtained. A band at 3626 cm(-1) attributed to the inner surface hydroxyls of kaolinite hydrogen bonded to hydrazine is observed. The intercalation of hydrazine-hydrate into kaolinite is complex and results from the different types of surface interactions of the hydrazine with the kaolinite surfaces.     

(Eta3-phenylallyl)(phosphanyloxazoline)palladium complexes: X-ray crystallographic studies,NMR investigations,and Ab initio/DFT calculations

All possible (eta(3)-allyl)palladium complexes (1-4) of the ligand (4S)-[2-(2'-diphenylphosphanyl)phenyl]-4,5-dihydro-4-(2-propyl)-oxazole (L 1) and eta(3)-allyl ligands with one to three phenyl substituents at the terminal allylic centers were synthesized and characterized by X-ray crystal structure analysis and, with respect to allylic isomers, by NMR investigations. Equilibrium geometries, electronic structures, and relative energies of isomeric complexes were computed by restricted Hartree-Fock (RHF) and density functional theory (DFT) calculations; experimentally determined isomer ratios could be reproduced. The results allowed important conclusions to be drawn regarding the mechanism of Pd-catalyzed asymmetric allylic substitutions.     

The dissociation kinetics of energy-selected CpMn(CO)(3)(+) ions studied by threshold photoelectron-photoion coincidence spectroscopy

Threshold photoelectron-photoion coincidence spectroscopy has been used to investigate the dissociation kinetics of the cyclopentadienyl manganese tricarbonyl ion, CpMn(CO)(3)(+). The ionization energy of CpMn(CO)(3) was measured from the threshold photoelectron spectrum to be 7.69 +/- 0.02 eV. The dissociation of the CpMn(CO)(3)(+) ion proceeds by the sequential loss of three CO molecules. The first and third CO loss reactions were observed to be slow (lifetimes in the microsecond range). By simulating the resulting asymmetric time-of-flight peak shapes and breakdown diagram, 0 K onsets for three product ions were determined to be 8.80 +/- 0.04, 9.43 +/- 0.04, and 10.51 +/- 0.06 eV, respectively. Combined with the adiabatic ionization energy, the three successive Mn-CO bond energies in the CpMn(CO)(3)(+) were found to be alternating with values of 1.11 +/- 0.04, 0.63 +/- 0.04, and 1.08 +/- 0.06 eV, respectively. Using a scaled theoretical Cp-Mn(+) bond energy of 3.10 +/- 0.10 eV and the combined results from theory and experiment, the 298 K gas-phase heat of formation of CpMn(CO)(3) is suggested to be -419 +/- 15 kJ/mol. Based on this value, the 298 K heats of formation of CpMn(CO)(3)(+), CpMn(CO)(2)(+), CpMnCO(+), and CpMn(+) are 325 +/- 15, 546 +/- 15, 719 +/- 15, and 938 +/- 15 kJ/mol, respectively. By scaling theoretical calculated neutral bond energies with the experimental information derived in this study, the successive Mn-CO bond energies were estimated to be 1.88, 1.10, and 1.03 eV, respectively, while the Cp-Mn bond energy was found to be 2.16 eV. Comparison between the quantum chemical calculations and experimental values shows that the loss of CO groups follows the lowest energy adiabatic path, in which electronic spin on the metal center is not conserved.