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Bert L. Vallee - One of the best experts on this subject based on the ideXlab platform.
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Zinc binding characteristics of the synthetic peptide corresponding to the structural Zinc site of horse liver alcohol dehydrogenase.
Advances in experimental medicine and biology, 1999Co-Authors: Tomas Bergman, Hans Jörnvall, Carina Palmberg, David S. Auld, Bert L. ValleeAbstract:Medium-chain dehydrogenases/reductases of the liver alcohol dehydrogenase type are Zinc metalloenzymes (Vallee and Hoch, 1957; IAeson, 1964; Drum et al., 1969), with two Zinc Atoms per subunit, one catalytic at the active site and one structural at a site influencing subunit interactions (Sytkowski and Vallee, 1976; Branden et al., 1975). In horse liver alcohol dehydrogenase and in all other mammalian liver forms, the structural Zinc Atom is liganded by four closely spaced Cys residues, at positions 97, 100, 103, and III (Branden et al., 1975; Vallee and Auld, 1990). The mechanism by which this Zinc Atom maintains its structural role is largely unknown. To probe the metal-binding characteristics of the structural Zinc site of alcohol dehydrogenase, we have analyzed Zinc-binding to a synthetic replica of the protein segment containing the four Cys residues and covering residues 93–115 of the parent molecule.
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A synthetic peptide encompassing the binding site of the second Zinc Atom (the 'structural' Zinc) of alcohol dehydrogenase.
European journal of biochemistry, 1992Co-Authors: Tomas Bergman, Hans Jörnvall, Barton Holmquist, Bert L. ValleeAbstract:A 23-residue peptide was synthesized that incorporates the loop which binds the structural Zinc Atom of mammalian alcohol dehydrogenases and contributes, in part, to subunit interactions in the native enzyme. Neither the amino acid composition nor the sequence of the peptide resemble those of Zinc fingers. The reduced peptide stoichiometrically binds Zinc or cobalt to form stable complexes with a dissociation constant for the peptide/Co2+ complex of 2.1 μM at pH 7.5. EDTA disrupts the complex. The absorption and magnetic circular dichroic spectra of the cobalt-peptide are indicative of a tetrahedral coordination geometry, and are similar to those of the cobalt-substituted structural site of horse and human (β1β1) liver alcohol dehydrogenases. Consequently, the synthetic peptide can serve as a model for the metal-binding segment of alcohol dehydrogenase and for studies of fundamental problems concerning protein/metal interactions.
Hans Jörnvall - One of the best experts on this subject based on the ideXlab platform.
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Zinc binding characteristics of the synthetic peptide corresponding to the structural Zinc site of horse liver alcohol dehydrogenase.
Advances in experimental medicine and biology, 1999Co-Authors: Tomas Bergman, Hans Jörnvall, Carina Palmberg, David S. Auld, Bert L. ValleeAbstract:Medium-chain dehydrogenases/reductases of the liver alcohol dehydrogenase type are Zinc metalloenzymes (Vallee and Hoch, 1957; IAeson, 1964; Drum et al., 1969), with two Zinc Atoms per subunit, one catalytic at the active site and one structural at a site influencing subunit interactions (Sytkowski and Vallee, 1976; Branden et al., 1975). In horse liver alcohol dehydrogenase and in all other mammalian liver forms, the structural Zinc Atom is liganded by four closely spaced Cys residues, at positions 97, 100, 103, and III (Branden et al., 1975; Vallee and Auld, 1990). The mechanism by which this Zinc Atom maintains its structural role is largely unknown. To probe the metal-binding characteristics of the structural Zinc site of alcohol dehydrogenase, we have analyzed Zinc-binding to a synthetic replica of the protein segment containing the four Cys residues and covering residues 93–115 of the parent molecule.
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Features of structural Zinc in mammalian alcohol dehydrogenase. Site-directed mutagenesis of the Zinc ligands.
European journal of biochemistry, 1994Co-Authors: Jana Jeloková, Hans Jörnvall, Christina Karlsson, Mats Estonius, Jan-olov HöögAbstract:All four cysteine ligands to the structural Zinc Atom of human class-I and class-III alcohol dehydrogenase have been exchanged by site-directed mutagenesis in order to study the importance of the metal in the mammalian enzymes. The cysteine residues were replaced with Ala and Ser, residues that are not able to ligand Zinc. All mutations resulted in inactive, unstable enzymes, in contrast to the non-mutated human alcohol dehydrogenases that are easily isolated. Northern-blot analysis revealed the presence of the expected mRNAs from expression plasmids constructed with the different mutated and non-mutated alcohol dehydrogenases, and Western-blot analysis gave faint signals for the mutated recombinant proteins from crude extracts. This verifies that the plasmid constructs are correct, but that the translated, mutated proteins lacking the Zinc-stabilized local fold, are subject to rapid degradation. Hence, the results directly illustrate the importance of the structural Zinc Atom in mammalian alcohol dehydrogenase and confirm it as a component with ‘structural’ properties. The results are compatible with those from sensitivities to proteases and from the structures of other proteins within the super-family, indicating that the structural role of the Zinc Atom may involve conservation of interfaces regulating the enzyme quaternary structure.
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A synthetic peptide encompassing the binding site of the second Zinc Atom (the 'structural' Zinc) of alcohol dehydrogenase.
European journal of biochemistry, 1992Co-Authors: Tomas Bergman, Hans Jörnvall, Barton Holmquist, Bert L. ValleeAbstract:A 23-residue peptide was synthesized that incorporates the loop which binds the structural Zinc Atom of mammalian alcohol dehydrogenases and contributes, in part, to subunit interactions in the native enzyme. Neither the amino acid composition nor the sequence of the peptide resemble those of Zinc fingers. The reduced peptide stoichiometrically binds Zinc or cobalt to form stable complexes with a dissociation constant for the peptide/Co2+ complex of 2.1 μM at pH 7.5. EDTA disrupts the complex. The absorption and magnetic circular dichroic spectra of the cobalt-peptide are indicative of a tetrahedral coordination geometry, and are similar to those of the cobalt-substituted structural site of horse and human (β1β1) liver alcohol dehydrogenases. Consequently, the synthetic peptide can serve as a model for the metal-binding segment of alcohol dehydrogenase and for studies of fundamental problems concerning protein/metal interactions.
Tomas Bergman - One of the best experts on this subject based on the ideXlab platform.
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Zinc binding characteristics of the synthetic peptide corresponding to the structural Zinc site of horse liver alcohol dehydrogenase.
Advances in experimental medicine and biology, 1999Co-Authors: Tomas Bergman, Hans Jörnvall, Carina Palmberg, David S. Auld, Bert L. ValleeAbstract:Medium-chain dehydrogenases/reductases of the liver alcohol dehydrogenase type are Zinc metalloenzymes (Vallee and Hoch, 1957; IAeson, 1964; Drum et al., 1969), with two Zinc Atoms per subunit, one catalytic at the active site and one structural at a site influencing subunit interactions (Sytkowski and Vallee, 1976; Branden et al., 1975). In horse liver alcohol dehydrogenase and in all other mammalian liver forms, the structural Zinc Atom is liganded by four closely spaced Cys residues, at positions 97, 100, 103, and III (Branden et al., 1975; Vallee and Auld, 1990). The mechanism by which this Zinc Atom maintains its structural role is largely unknown. To probe the metal-binding characteristics of the structural Zinc site of alcohol dehydrogenase, we have analyzed Zinc-binding to a synthetic replica of the protein segment containing the four Cys residues and covering residues 93–115 of the parent molecule.
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A synthetic peptide encompassing the binding site of the second Zinc Atom (the 'structural' Zinc) of alcohol dehydrogenase.
European journal of biochemistry, 1992Co-Authors: Tomas Bergman, Hans Jörnvall, Barton Holmquist, Bert L. ValleeAbstract:A 23-residue peptide was synthesized that incorporates the loop which binds the structural Zinc Atom of mammalian alcohol dehydrogenases and contributes, in part, to subunit interactions in the native enzyme. Neither the amino acid composition nor the sequence of the peptide resemble those of Zinc fingers. The reduced peptide stoichiometrically binds Zinc or cobalt to form stable complexes with a dissociation constant for the peptide/Co2+ complex of 2.1 μM at pH 7.5. EDTA disrupts the complex. The absorption and magnetic circular dichroic spectra of the cobalt-peptide are indicative of a tetrahedral coordination geometry, and are similar to those of the cobalt-substituted structural site of horse and human (β1β1) liver alcohol dehydrogenases. Consequently, the synthetic peptide can serve as a model for the metal-binding segment of alcohol dehydrogenase and for studies of fundamental problems concerning protein/metal interactions.
Chaitali Mukhophadhyay - One of the best experts on this subject based on the ideXlab platform.
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Role of Zinc in tRNA-acceptor Stem Binding by Glutamyl-tRNA Synthetase From E.coli: A Molecular Modeling Study
Journal of biomolecular structure & dynamics, 1997Co-Authors: Asim K. Bothra, Siddhartha Roy, Chhabinath Mandal, Chaitali MukhophadhyayAbstract:A model of the N-terminal half of glutamyl-tRNA synthetase from E. coli was constructed on the basis of similarity in sequence and function of Glutaminyl- and Glutamyl-tRNA synthetases. The glutaminyl-tRNA synthetase does not contain any Zinc Atom, but glutamyl-tRNA synthetase from E. coli contains one Atom of Zinc. The specific role of Zinc is not yet known. In this article, molecular modeling is employed to show that the Zinc Atom is well outside the contact region of the acceptor stem of tRNA. The placement of a Zinc Atom at a significant distance from the tRNA acceptor stem indicates that the role of Zinc is likely to be indirect and structural.
Sylvain Blanquet - One of the best experts on this subject based on the ideXlab platform.
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Mapping of the Zinc binding domain of Escherichia coli methionyl-tRNA synthetase
Journal of molecular biology, 1993Co-Authors: Dominique Fourmy, Thierry Meinnel, Yves Mechulam, Sylvain BlanquetAbstract:Abstract Cys/His motifs, found in several nucleic acid binding proteins, generally correspond to sites for the binding of metal Atoms. Such a motif, comprising four Cys residues, occurs in the subunits of Escherichia coli methionyl-tRNA synthetase, a dimeric enzyme known to bind two Zinc Atoms. In this study, each of the four cysteines in the cysteine cluster (region 145 to 161) of E. coli methionyl-tRNA synthetase were successively changed into an alanine. Either substitution is sufficient to destabilize the tight binding of the Zinc ion. Moreover, a peptide having a sequence corresponding to that of the 138 to 163 region of methionyl-tRNA synthetase has been prepared. It strongly binds one Zinc Atom, even in the presence of ethylene diamine tetraacetate. These data establish that, in methionyl-tRNA synthetase, the Cys motif of region 145 to 161 is actually the binding site for Zinc. In addition, the mutation of each cysteine modifies the parameters of the methionine activation reaction, and appears to change the structure of the enzyme, as probed by an increased sensitivity of the mutant enzymes to trypsin attack. The possible role of the Zinc Atom and of its chelating residues in the folding of the active centre of methionyl-tRNA synthetase is discussed.
