ABSTRACT
Cu(A) is an electron-transfer copper center present in heme-copper oxidases and N2O reductases. The center is a binuclear unit, with two cysteine ligands bridging the metal ions and two terminal histidine residues. A Met residue and a peptide carbonyl group are located on opposite sides of the Cu2S2 plane; these weaker ligands are fully conserved in all known Cu(A) sites. The Met160Gln mutant of the soluble subunit II of Thermus thermophilus ba3 oxidase has been studied by NMR spectroscopy. In its oxidized form, the binuclear copper is a fully delocalized mixed-valence pair, as are all natural Cu(A) centers. The faster nuclear relaxation in this mutant suggests that a low-lying excited state has shifted to higher energies compared to that of the wild-type protein. The introduction of the Gln residue alters the coordination mode of His114 but does not affect His157, thereby confirming the proposal that the axial ligand-to-copper distances influence the copper-His interactions (Robinson, H.; Ang, M. C.; Gao, Y. G.; Hay, M. T.; Lu, Y.; Wang, A. H. Biochemistry 1999, 38, 5677). Changes in the hyperfine coupling constants of the Cys beta-CH2 groups are attributed to minor geometrical changes that affect the Cu-S-C(beta)-H(beta) dihedral angles. These changes, in addition, shift the thermally accessible excited states, thus influencing the spectral position of the Cys beta-CH2 resonances. The Cu-Cys bonds are not substantially altered by the Cu-Gln160 interaction, in contrast to the situation found in the evolutionarily related blue copper proteins. It is possible that regulatory subunits in the mitochondrial oxidases fix the relative positions of thermally accessible Cu(A) excited states by tuning axial ligand interactions.
Subject(s)
Copper/chemistry , Cytochrome b Group/chemistry , Electron Transport Complex IV/chemistry , Cytochrome b Group/genetics , Electron Transport Complex IV/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Glutamine/chemistry , Glutamine/genetics , Methionine/chemistry , Methionine/genetics , Models, Molecular , Mutagenesis , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Conformation , Protein Structure, Tertiary , Protons , Thermus thermophilus/enzymology , Thermus thermophilus/geneticsABSTRACT
The structural and functional features of class B b-lactamases, which are metal-dependent, are reviewed in this article. Enzymes from different bacterial strains exhibit a common fold and sequence similarity in their active sites. However, the protein scaffold fine tunes the metal binding affinity and substrate selectivity. In this way, some metallo-b-lactamases seem to be functional with only one Zn(II) equivalent per enzyme, whereas others require a binuclear active site. The sequence similarity leads to a subdivision of these enzymes into three subclasses. The substrate specificities are rather broad, except for enzymes belonging to subclass B2. Some inhibitors have been designed and tested, but none of them is able to exhibit a broad spectrum against these enzymes.
Subject(s)
Metalloproteins/antagonists & inhibitors , Metalloproteins/chemistry , Zinc/chemistry , beta-Lactamase Inhibitors , beta-Lactamases/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Base Sequence , Binding Sites , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Hydrolysis , Metalloproteins/classification , Models, Molecular , Molecular Sequence Data , beta-Lactamases/classification , beta-LactamsABSTRACT
The zinc metalloenzyme beta-lactamase II (betaLII) from Bacillus cereus has been overexpressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and the metal binding properties of recombinant betaLII toward Zn(II) and Co(II) have been studied by fluorescence and activity measurements. The apoenzyme is able to bind two metal ion equivalents, which confer on betaLII its maximum enzymatic efficiency. The enzyme is partially active with one metal ion equivalent. The diCo(II) and a mixed Zn(II)Co(II) derivative of betaLII were obtained and probed by electronic and paramagnetic NMR spectroscopy. In the high-affinity site, the metal is bound to three His residues and a solvent molecule, adopting a tetrahedral geometry. A Cys, a His, and an Asp residue are coordinated to the low-affinity metal site, together with two or three solvent molecules. This coordination polyhedron resembles the binuclear metal site of the Bacteroides fragilis beta-lactamase [Concha, N., Rasmussen, B. A., Bush, K., and Herzberg, O. (1996) Structure 4, 823-836; Carfi, A., Duée, E., Paul-Soto, R., Galleni, M., Frère, J. M., and Dideberg, O. (1998) Acta Crystallogr. D54, 47-57] but differs from that resulting from the X-ray study of betaLII [Carfi, A., Pares, S., Duée, E., Galleni, M., Duez, C., Frère, J. M., and Dideberg, O. (1995) EMBO J. 14, 4914-4921]. These results suggest that this binuclear metal site may be a general feature of metallo-beta-lactamases.
Subject(s)
Bacillus cereus/enzymology , Cephalosporinase/chemistry , Zinc/chemistry , Amino Acid Sequence , Binding Sites , Cephalosporinase/biosynthesis , Cephalosporinase/genetics , Cobalt/chemistry , Conserved Sequence , Escherichia coli/genetics , Magnetic Resonance Spectroscopy , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Spectrometry, Fluorescence , SpectrophotometryABSTRACT
The triflates and pivalates of 3 alpha-hydroxymethyl-6-substituted-2,2-dimethylpenam sulfones 3, 5; methyl and benzyl 6-substituted penicillanates 6-9 and 3-exo-methylene-6-substituted-2,2-dimethylpenam sulfone 4 were synthesized. These novel compounds were evaluated as elastase inhibitors using porcine pancreatic elastase. The effects that structural modifications of substituents on C-3 and C-6 in the penam nucleus have on elastase activity were examined and several similarities and distinctions were identified when compared to the reported penicillin esters and amides elastase inhibitors.