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Identification and characterization of the functional amino acids at the active center of pig liver thioltransferase by site-directed mutagenesis.

Identifieur interne : 001303 ( Main/Exploration ); précédent : 001302; suivant : 001304

Identification and characterization of the functional amino acids at the active center of pig liver thioltransferase by site-directed mutagenesis.

Auteurs : Y F Yang [États-Unis] ; W W Wells

Source :

RBID : pubmed:2061338

Descripteurs français

English descriptors

Abstract

By using site-directed mutagenesis techniques, the essential amino acids at the catalytic center of porcine thioltransferase (glutaredoxin) were determined. Seven oligonucleotides were designed, synthesized, and used to construct mutants, ETT-C22S, ETT-C25S, ETT-C25A, ETT-R26V, ETT-K27Q, ETT-R26V: K27Q, and ETT-C78S:C82S, by altering their codons in pig liver thioltransferase cDNA/M13mp18 clones. Each of the thioltransferases was purified to homogeneity and its dithiol-disulfide exchange, and dehydroascorbate reductase activities were compared with those of the wild-type (ETT). Evidence was obtained that Cys22 was essential for catalytic activity, and the extremely low pKa value of its sulfhydryl group was facilitated primarily by Arg26. The role of Lys27 at the active center was different from that of Arg26 and may be important in stabilizing the E.S intermediate by electrostatic forces. The second pair of cysteines, Cys78 and Cys82, nearer the C terminus, were not directly involved in the active center, but may play a role in defining the native protein structure. The replacement of the original Cys with a Ser at position 25 increased rather than decreased the enzyme activity, suggesting that the proposed intramolecular disulfide bond between Cys22 and Cys25 is not necessary for the catalytic mechanism of the Ser25 mutant, but does not rule out such a mechanism for the wild-type enzyme.

PubMed: 2061338


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Le document en format XML

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<title xml:lang="en">Identification and characterization of the functional amino acids at the active center of pig liver thioltransferase by site-directed mutagenesis.</title>
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<name sortKey="Yang, Y F" sort="Yang, Y F" uniqKey="Yang Y" first="Y F" last="Yang">Y F Yang</name>
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<nlm:affiliation>Department of Biochemistry, Michigan State University, East Lansing 48824.</nlm:affiliation>
<orgName type="university">Université d'État du Michigan</orgName>
<country>États-Unis</country>
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<name sortKey="Wells, W W" sort="Wells, W W" uniqKey="Wells W" first="W W" last="Wells">W W Wells</name>
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<term>Amino Acids (genetics)</term>
<term>Amino Acids (isolation & purification)</term>
<term>Animals (MeSH)</term>
<term>Base Sequence (MeSH)</term>
<term>Binding Sites (MeSH)</term>
<term>Blotting, Western (MeSH)</term>
<term>Electrophoresis, Polyacrylamide Gel (MeSH)</term>
<term>Escherichia coli (genetics)</term>
<term>Gene Expression Regulation, Bacterial (MeSH)</term>
<term>Gene Expression Regulation, Enzymologic (MeSH)</term>
<term>Genes, Bacterial (MeSH)</term>
<term>Glutaredoxins (MeSH)</term>
<term>Hydrogen-Ion Concentration (MeSH)</term>
<term>Isoelectric Focusing (MeSH)</term>
<term>Kinetics (MeSH)</term>
<term>Liver (enzymology)</term>
<term>Molecular Sequence Data (MeSH)</term>
<term>Mutagenesis, Site-Directed (MeSH)</term>
<term>Oxidoreductases (genetics)</term>
<term>Oxidoreductases (metabolism)</term>
<term>Protein Disulfide Reductase (Glutathione) (MeSH)</term>
<term>Swine (MeSH)</term>
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<term>Acides aminés (composition chimique)</term>
<term>Acides aminés (génétique)</term>
<term>Acides aminés (isolement et purification)</term>
<term>Animaux (MeSH)</term>
<term>Cinétique (MeSH)</term>
<term>Concentration en ions d'hydrogène (MeSH)</term>
<term>Données de séquences moléculaires (MeSH)</term>
<term>Escherichia coli (génétique)</term>
<term>Focalisation isoélectrique (MeSH)</term>
<term>Foie (enzymologie)</term>
<term>Glutarédoxines (MeSH)</term>
<term>Gènes bactériens (MeSH)</term>
<term>Mutagenèse dirigée (MeSH)</term>
<term>Oxidoreductases (génétique)</term>
<term>Oxidoreductases (métabolisme)</term>
<term>Protein-disulfide reductase (glutathione) (MeSH)</term>
<term>Régulation de l'expression des gènes bactériens (MeSH)</term>
<term>Régulation de l'expression des gènes codant pour des enzymes (MeSH)</term>
<term>Sites de fixation (MeSH)</term>
<term>Suidae (MeSH)</term>
<term>Séquence d'acides aminés (MeSH)</term>
<term>Séquence nucléotidique (MeSH)</term>
<term>Technique de Western (MeSH)</term>
<term>Électrophorèse sur gel de polyacrylamide (MeSH)</term>
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<term>Escherichia coli</term>
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<term>Acides aminés</term>
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<term>Oxidoreductases</term>
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<term>Amino Acid Sequence</term>
<term>Animals</term>
<term>Base Sequence</term>
<term>Binding Sites</term>
<term>Blotting, Western</term>
<term>Electrophoresis, Polyacrylamide Gel</term>
<term>Gene Expression Regulation, Bacterial</term>
<term>Gene Expression Regulation, Enzymologic</term>
<term>Genes, Bacterial</term>
<term>Glutaredoxins</term>
<term>Hydrogen-Ion Concentration</term>
<term>Isoelectric Focusing</term>
<term>Kinetics</term>
<term>Molecular Sequence Data</term>
<term>Mutagenesis, Site-Directed</term>
<term>Protein Disulfide Reductase (Glutathione)</term>
<term>Swine</term>
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<term>Concentration en ions d'hydrogène</term>
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<term>Focalisation isoélectrique</term>
<term>Glutarédoxines</term>
<term>Gènes bactériens</term>
<term>Mutagenèse dirigée</term>
<term>Protein-disulfide reductase (glutathione)</term>
<term>Régulation de l'expression des gènes bactériens</term>
<term>Régulation de l'expression des gènes codant pour des enzymes</term>
<term>Sites de fixation</term>
<term>Suidae</term>
<term>Séquence d'acides aminés</term>
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<front>
<div type="abstract" xml:lang="en">By using site-directed mutagenesis techniques, the essential amino acids at the catalytic center of porcine thioltransferase (glutaredoxin) were determined. Seven oligonucleotides were designed, synthesized, and used to construct mutants, ETT-C22S, ETT-C25S, ETT-C25A, ETT-R26V, ETT-K27Q, ETT-R26V: K27Q, and ETT-C78S:C82S, by altering their codons in pig liver thioltransferase cDNA/M13mp18 clones. Each of the thioltransferases was purified to homogeneity and its dithiol-disulfide exchange, and dehydroascorbate reductase activities were compared with those of the wild-type (ETT). Evidence was obtained that Cys22 was essential for catalytic activity, and the extremely low pKa value of its sulfhydryl group was facilitated primarily by Arg26. The role of Lys27 at the active center was different from that of Arg26 and may be important in stabilizing the E.S intermediate by electrostatic forces. The second pair of cysteines, Cys78 and Cys82, nearer the C terminus, were not directly involved in the active center, but may play a role in defining the native protein structure. The replacement of the original Cys with a Ser at position 25 increased rather than decreased the enzyme activity, suggesting that the proposed intramolecular disulfide bond between Cys22 and Cys25 is not necessary for the catalytic mechanism of the Ser25 mutant, but does not rule out such a mechanism for the wild-type enzyme.</div>
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<AbstractText>By using site-directed mutagenesis techniques, the essential amino acids at the catalytic center of porcine thioltransferase (glutaredoxin) were determined. Seven oligonucleotides were designed, synthesized, and used to construct mutants, ETT-C22S, ETT-C25S, ETT-C25A, ETT-R26V, ETT-K27Q, ETT-R26V: K27Q, and ETT-C78S:C82S, by altering their codons in pig liver thioltransferase cDNA/M13mp18 clones. Each of the thioltransferases was purified to homogeneity and its dithiol-disulfide exchange, and dehydroascorbate reductase activities were compared with those of the wild-type (ETT). Evidence was obtained that Cys22 was essential for catalytic activity, and the extremely low pKa value of its sulfhydryl group was facilitated primarily by Arg26. The role of Lys27 at the active center was different from that of Arg26 and may be important in stabilizing the E.S intermediate by electrostatic forces. The second pair of cysteines, Cys78 and Cys82, nearer the C terminus, were not directly involved in the active center, but may play a role in defining the native protein structure. The replacement of the original Cys with a Ser at position 25 increased rather than decreased the enzyme activity, suggesting that the proposed intramolecular disulfide bond between Cys22 and Cys25 is not necessary for the catalytic mechanism of the Ser25 mutant, but does not rule out such a mechanism for the wild-type enzyme.</AbstractText>
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