The Protein Conformation of Cd‐Substituted Horse Liver Alcohol Dehydrogenase and its Metal‐Site Coordination Geometry in Binary and Ternary Inhibitor Complexes

Hemmingsen, Lars; Bauer, Rogert; Bjerrum, Morten J.; Adolph, Hans-Werner; Zeppezauer, Michael; Cedergren-Zeppezauer, Eila

doi:10.1111/j.1432-1033.1996.00546.x

Cited by 27 publications

(38 citation statements)

References 37 publications

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“…PAC Spectroscopy. PAC spectroscopy and sample preparation were performed as described (35,36); however, a typing error of ''10 -40 mL'' should be ''10 -40 L,'' and the current experiment with 111m Cd and TRIL12LDL16C was carried out in a 1:12 ratio, at pH 9.1, 1°C, and in 55% wt/wt sucrose to further reduce the Brownian tumbling of the molecules. Finally, all fits were carried out with 300 data points, disregarding the 5 first points due to systematic errors in these.…”

Section: Methodsmentioning

confidence: 99%

Using diastereopeptides to control metal ion coordination in proteins

Peacock

Hemmingsen

Pecoraro

2008

Proc. Natl. Acad. Sci. U.S.A.

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Here, we report a previously undescribed approach for controlling metal ion coordination geometry in biomolecules by reorientating amino acid side chains through substitution of L-to D-amino acids. These diastereopeptides allow us to manipulate the spatial orientation of amino acid side chains to alter the sterics of metal binding pockets. We have used this approach to design the de novo metallopeptide, Cd (TRIL12L DL16C)

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Section: Methodsmentioning

confidence: 99%

Using diastereopeptides to control metal ion coordination in proteins

Peacock

Hemmingsen

Pecoraro

2008

Proc. Natl. Acad. Sci. U.S.A.

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“…The relation between these two parameters and the frequencies in G 2 (t) can be found in Ref. 23. Thus, from the time dependence of G 2 (t), 0 and , determined through least squares fitting, reflect the coordination geometry of the cadmium ion.…”

Section: Perturbed Angular Correlation Of ␥-Rays Spectroscopymentioning

confidence: 99%

“…Preparation and purification of 111m Cd is described in Ref. 23. The PAC spectrometer is described in Ref.…”

Section: Perturbed Angular Correlation Of ␥-Rays Spectroscopymentioning

confidence: 99%

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Coordination Geometries of Metal Ions in D- or L-Captopril-inhibited Metallo-β-lactamases

Heinz

Bauer²,

Wommer

et al. 2003

Journal of Biological Chemistry

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D-and L-captopril are competitive inhibitors of metallo-␤-lactamases. For the enzymes from Bacillus cereus (BcII) and Aeromonas hydrophila (CphA), we found that the mononuclear enzymes are the favored targets for inhibition. By combining results from extended x-ray absorption fine structure, perturbed angular correlation of ␥-rays spectroscopy, and a study of metal ion binding, we derived that for Cd(II) 1 -BcII, the thiolate sulfur of D-captopril binds to the metal ion located at the site defined by three histidine ligand residues. This is also the case for the inhibited Co(II) 1 and Co(II) 2 enzymes as observed by UV-visible spectroscopy. Although the single metal ion in Cd(II) 1 -BcII is distributed between both available binding sites in both the uninhibited and the inhibited enzyme, Cd(II) 1 -CphA shows only one defined ligand geometry with the thiolate sulfur coordinating to the metal ion in the site composed of 1 Cys, 1 His, and 1 Asp. CphA shows a strong preference for D-captopril, which is also reflected in a very rigid structure of the complex as determined by perturbed angular correlation spectroscopy. For BcII and CphA, which are representatives of the metallo-␤-lactamase subclasses B1 and B2, we find two different inhibitor binding modes.Metallo-␤-lactamases confer antibiotic resistance to bacteria by catalyzing the hydrolysis of ␤-lactam antibiotics, including carbapenems. This relatively new form of resistance is spreading and thereby escaping the effective inhibitors developed to fight the better known serine-␤-lactamases. For all metallo-␤-lactamases investigated, structurally similar enzyme active sites comprising two zinc binding sites are reported. For Bacillus cereus metallo-␤-lactamase (BcII), 1 one metal-binding site contains three His (H-site); the other one contains 1 Asp, 1 Cys, and 1 His as the metal ligating residues (DCH site) as derived from x-ray crystallography (1). For CphA, 1 His from the H-site (His-116) is supposed to be replaced by an Asn (2). Various thiol-carboxylate compounds were identified as potent inhibitors (3). The active site binding of thiomandelic acid to BcII was studied by NMR spectroscopy (4), whereas the binding of 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mercaptomethyl)-4-(phenylbutrylglycine)] to the enzyme from Pseudomonas aeruginosa (IMP-1) was characterized by x-ray crystallography (5). With both approaches, the inhibited binuclear zinc enzymes were studied. Both studies agree in a bridging role of the metal-bound sulfur of the inhibitor, whereas the carboxylate group of the inhibitors binds to an accessible amino acid, thus stabilizing the complex. Other known inhibitory compounds are 2,3-(S,S)-disubstituted succinic acids for IMP-1 (6) or moxalactam and cefoxitin for CphA (7). The latter compounds lead to irreversible inactivation of the enzyme by the hydrolyzed reaction products.The structural investigation of D-and L-captopril binding presented here is based on results obtained from enzyme kinetic and thermodynamic studies. Captopril is known as an ...

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“…Preparation and purification of 111m Cd is described in Ref. 19. The PAC spectrometer is described in Ref.…”

Section: Site-directed Mutagenesis Production and Purification Of Ementioning

confidence: 99%

Metal Ion Binding and Coordination Geometry for Wild Type and Mutants of Metallo-β-lactamase from Bacillus cereus569/H/9 (BcII)

Seny¹,

Heinz²,

Wommer³

et al. 2001

Journal of Biological Chemistry

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117

174

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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...

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The Protein Conformation of Cd‐Substituted Horse Liver Alcohol Dehydrogenase and its Metal‐Site Coordination Geometry in Binary and Ternary Inhibitor Complexes

Cited by 27 publications

References 37 publications

Using diastereopeptides to control metal ion coordination in proteins

Using diastereopeptides to control metal ion coordination in proteins

Coordination Geometries of Metal Ions in D- or L-Captopril-inhibited Metallo-β-lactamases

Metal Ion Binding and Coordination Geometry for Wild Type and Mutants of Metallo-β-lactamase from Bacillus cereus569/H/9 (BcII)

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