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1.
J Biol Inorg Chem ; 9(5): 550-62, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15156371

ABSTRACT

The inherent ability to interact with DNA makes cationic metallo-porphyrins attractive targets as antitumor drugs. Many studies describe their interaction with DNA and the mechanism by which they induce DNA cleavage. Since porphyrins can be used as anchors for chemically reactive groups, it is possible to modify them to generate a family of compounds with specific functions. In the present work, we used chemical groups such as copper-bipyridinium (Cu-bpy), which hydrolyze phosphodiester bonds, and a porphyrin core to synthesize two novel Cu(2)-bpy-porphyrins. Their interactions with DNA have been characterized using classic spectroscopic methods, and their oxidative and hydrolytic reactivity toward supercoiled plasmid DNA has been studied in vitro. Our results show that Cu(2)-bpy-porphyrins interact with DNA via external association and intercalation and that their ability to cleave DNA and the mechanisms depends on the experimental conditions.


Subject(s)
2,2'-Dipyridyl/chemistry , Copper/chemistry , DNA/chemistry , Porphyrins/chemical synthesis , 2,2'-Dipyridyl/metabolism , Anaerobiosis , Cations , Cerium/chemistry , Circular Dichroism , Copper/metabolism , DNA/metabolism , Electrophoresis, Agar Gel , Free Radical Scavengers , Hydrolysis , Intercalating Agents/chemistry , Models, Chemical , Oxidation-Reduction , Porphyrins/metabolism , Spectrophotometry, Ultraviolet , Temperature
2.
Inorg Chem ; 39(12): 2666-75, 2000 Jun 12.
Article in English | MEDLINE | ID: mdl-11197024

ABSTRACT

The problem of metal selectivity of iron/manganese superoxide dismutases (SODs) is addressed through the electronic structures of active sites using electron paramagnetic resonance and ligand field calculations. Studies of wild-type iron(III) SOD (FeSOD) from Escherichia coli and from Methanobacterium thermoautotrophicum and iron-substituted manganese(III) SOD (Fe(sub)MnSOD) from E. coli and from Serratia marcescens are reported. EPR spectroscopy of wild-type enzymes shows transitions within all three Kramers doublets identified by their g values. From the temperature dependence of the observed transitions, the zero-field splitting is found to be negative, D = -2 +/- 0.2 cm-1. The electronic structure is typical of a distorted trigonal bipyramid, all the EPR features being reproduced by ligand field analysis. This unique and necessary electronic structure characterizes wild-type enzymes whatever their classification from the amino acid sequence into iron or manganese types, as E. coli FeSOD or M. thermoautotrophicum FeSOD. In iron-substituted manganese SODs, reduced catalytic activity is found. We describe how inhomogeneity of all reported substituted MnSODs might explain the activity decrease. EPR spectra of substituted enzymes show several overlapping components. From simulation of these spectra, one component is identified which shares the same electronic structure of the wild-type FeSODs, with the proportion depending on pH. Ligand field calculations were performed to investigate distortions of the active site geometry which induce variation of the excitation energy of the lowest quartet state. The corresponding coupling between the ground state and the excited state is found to be maximum in the geometry of the native SODs. We conjecture that such coupling should be considered in the electron-transfer process and in the contribution of the typical electronic structure of FeSOD to the activity.


Subject(s)
Superoxide Dismutase/chemistry , Binding Sites , Electron Spin Resonance Spectroscopy , Iron/chemistry , Ligands , Manganese/chemistry , Molecular Structure
3.
FEBS Lett ; 401(1): 15-9, 1997 Jan 13.
Article in English | MEDLINE | ID: mdl-9003797

ABSTRACT

Manganese containing superoxide dismutase from E. coli has been investigated through paramagnetic NMR spectroscopy. The spectrum of the native form was rationalized using a tau(s) = 3x10(-11) s for the Mn(III) ion, consistent with previous estimates from NMRD measurements. Mn(III) has been replaced by a Co(II) ion and a tentative assignment of the NMR spectrum of the Co(II)-substituted derivative has been proposed, based on T1, chemical shifts and 1D-NOE data. The metal coordination geometry is provided by three histidines and a carboxylate group. The presence of a solvent molecule as a loosely bound fifth ligand is also proposed. The NMR data of the Co(II)-substituted derivative of E. coli MnSOD differs from those of Co(II)SOD from other bacterial sources. This suggests that Co(II) substitution is an efficient method to address the problem of metal ion selectivity in superoxide dismutase.


Subject(s)
Escherichia coli/enzymology , Superoxide Dismutase/chemistry , Magnetic Resonance Spectroscopy , Protein Conformation
4.
J Inorg Biochem ; 40(2): 127-39, 1990 Oct.
Article in English | MEDLINE | ID: mdl-2092077

ABSTRACT

The stability constants of a series of complexes of the cationic water-soluble porphyrin ZnTMPyP with various amino acids have been determined by 1H NMR spectroscopy at pH 10.5. The following stability order has been observed: Tyr greater than Phe, Glu greater than Asp greater than Ile greater than Val greater than Gly. These results can be best rationalized by invoking complex stabilization due to ligand-ligand (e.g., stacking or electrostatic) interactions. Evidence for stacking interactions between the porphyrin ring and the aromatic ring of phenylalanine, tyrosine, and tryptophan was further provided by study of the complexation of these amino acids with the free-base porphyrin TMPyPH2. In this case, complexation constants increased in the order: Phe less than Tyr less than Trp. Attempts to form complexes of the amino acids with the anionic porphyrin ZnTCPP proved unsuccessful, indicating that electrostatic interactions play a major role in the stability of the zinc porphyrin-amino acids complexes.


Subject(s)
Amino Acids/metabolism , Metalloporphyrins/metabolism , Zinc/metabolism , Amino Acids/chemistry , Anions , Cations , Chemical Phenomena , Chemistry, Physical , Drug Stability , Electrochemistry , Magnetic Resonance Spectroscopy , Metalloporphyrins/chemistry , Molecular Structure , Phenylalanine/metabolism , Protein Conformation , Tryptophan/analogs & derivatives , Tryptophan/metabolism , Tyrosine/metabolism , Zinc/chemistry
5.
Presse Med ; 15(46): 2303-8, 1986 Dec 20.
Article in French | MEDLINE | ID: mdl-2949272

ABSTRACT

Like all penicillins, piperacillin exhibits some susceptibility to beta-lactamases of the penicillinase type, including those produced by Staphylococcus aureus and the TEM, OXA and CARB enzymes isolated from Gram-negative bacilli. Piperacillin is very slightly hydrolyzed by cephalosporinases, which makes it similar to 3rd generation cephalosporins. When tested with Enterobacter cloacae GN 5797 and Pseudomonas aeruginosa NTC 8303, two strains which produce inducible cephalosporinases, piperacillin had moderate inductive activity compared to cefoxitin, a potent inducer. Induction was very low with Enterobacter, but the drug was slightly more sensitive to Pseudomonas. Inhibition of this type of beta-lactamase synthesis was very strong when piperacillin was combined with amikacin and weaker when it was combined with pefloxacin. The piperacillin-amikacin combination prevented the development of piperacillin-resistant mutants of Enterobacter and Pseudomonas and, probably, of all Gram-negative bacilli. In our tests, the piperacillin-pefloxacin combination was of interest only against Enterobacter, and probably against all enterobacteria, since Pseudomonas mutants that resist pefloxacin are fairly easily obtained in vitro.


Subject(s)
Piperacillin/pharmacology , beta-Lactamases/biosynthesis , Amikacin/pharmacology , Anti-Bacterial Agents/pharmacology , Drug Therapy, Combination , Enterobacter/metabolism , Enzyme Induction , Hydrolysis , Norfloxacin/analogs & derivatives , Norfloxacin/pharmacology , Pefloxacin , Piperacillin/metabolism , Pseudomonas aeruginosa/metabolism
6.
Ann Inst Pasteur Microbiol (1985) ; 137B(1): 19-27, 1986.
Article in English | MEDLINE | ID: mdl-3324941

ABSTRACT

A five-step procedure has been developed for purifying Pitton's type 2 plasmid-mediated beta-lactamase (PIT-2, also called SHV-1) from cultures of a hyperproducing variant of an Escherichia coli K12 strain carrying the plasmid p453. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a single protein band with a molecular weight of about 27,500. The amino acid composition of the protein and the amino acid sequence of the NH2-terminal region have been determined. PIT-2 enzyme contains 272 amino acid residues with 2 cysteines. Studies of the S-carboxymethylated protein (previously reduced or unreduced) suggest that these two residues are presumably in the form of free sulphydryl groups in the native protein. Conversely, TEM-2 beta-lactamase contains 2 cysteine residues which are in the form of a disulphide bond. Comparison of PIT-2 with other beta-lactamases was made using the difference index (DI) of Metzger et al. The PIT-2 enzyme appeared more closely related to the TEM-type penicillinases (DI of 6.5).


Subject(s)
Escherichia coli/enzymology , beta-Lactamases , Amino Acid Sequence , Amino Acids/analysis , Escherichia coli/genetics , Methods , Molecular Sequence Data , Plasmids , beta-Lactamases/analysis , beta-Lactamases/isolation & purification
7.
Am J Med ; 77(6A): 25-7, 1984 Dec 21.
Article in English | MEDLINE | ID: mdl-6097119

ABSTRACT

The interactions of cefmenoxime with beta-lactamases in comparison with cefotaxime, moxalactam, cefoperazone, and ceftazidime have been determined. On-line computerized microacidimetry allowed determination of the affinity of these compounds with the enzymes, which was characterized by Km values. The beta-lactamases that were used were two cephalosporinases and one penicillinase. Within these data, the cephalosporins could be classified into three groups: (1) those with high affinity for the cephalosporinases and very poor affinity for the penicillinase (cefmenoxime, cefotaxime, and moxalactam); (2) those with moderate affinity for the cephalosporinases and very poor affinity for the penicillinase (ceftazidime); (3) those with poor affinity for all enzymes (cefoperazone). In the case of the penicillinase (TEM-1), only cefoperazone was subject to some hydrolysis.


Subject(s)
Cefotaxime/analogs & derivatives , beta-Lactamases/metabolism , Bacteria/enzymology , Cefmenoxime , Cefotaxime/metabolism , Cephalosporinase/metabolism , Cephalosporins/metabolism , Hydrolysis , Kinetics , Moxalactam/metabolism , Penicillinase/metabolism
9.
J Antimicrob Chemother ; 11 Suppl: 147-52, 1983 Jan.
Article in English | MEDLINE | ID: mdl-6601654

ABSTRACT

Cefotetan is a new cephamycin whose interaction with a group of eight beta-lactamases isolated from various Gram-negative bacteria has been studied. Using computerized micro-acidimetry, cefotetan was found to be resistant to the hydrolytic action of various enzymes, including those which actively hydrolyse cefotaxime. Despite this resistance to hydrolysis, cefotetan nevertheless shows a certain affinity for these enzymes. These data were submitted to two statistical procedures consisting of correspondence analysis and ascending hierarchical classification. These techniques revealed that cefotetan behaves very much like cefoxitin (another cephamycin) and also like cefotaxime and moxalactam.


Subject(s)
Anti-Bacterial Agents , Cephalosporins , Cephamycins , beta-Lactamases , Cefotetan , Computers , Hydrolysis , Kinetics
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