| Differential reactivity of the functional sulfhydryl groups of cysteine-32 and cysteine-35 present in the reduced form of thioredoxin from Escherichia coli. | |
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MedLine Citation:
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PMID: 7000775 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Only one of the sulfhydryl groups from Cys-32 and Cys-35 in the active center of native Escherichia coli thioredoxin-(SH)2 was alkylated by excess iodoacetic acid at pH values below 8.0. Both groups reacted in the protein denatured with 4.5 M guanidine hydrochloride. The second order rate of alkylation of thioredoxin-(SH)2 with 1 eq of iodoacetic acid was pH-dependent and showed independent initial reactions of one thiolate ion with a pK value of 6.7 and a second with a pK value close to 9.0. The same pH dependence was observed for alkylation with iodoacetamide but the apparent rate constant, 107 M-1 S-1 at pH 7.2, was about 20-fold higher than the corresponding rate with iodoacetate. The sulfhydryl group with a pK value of 6.7 was shown to belong to Cys-32 by labeling thioredoxin with [14C]iodoacetic acid followed by complete alkylation with [3H]iodoacetate and amino acid sequence analysis of peptides from the active center. The abnormally low pK value of Cys-32 is suggested to arise by electrostatic influence from a positive charge on the amino group of Lys-36. A mechanism of action for thioredoxin-(SH)2 as a protein disulfide reductase has been formulated. This is based on an initial nucleophilic attack by the thiolate of Cys-32 with the formation of an unstable transient mixed disulfide involving Cys-32 and one of the sulfurs in the substrate. This is followed by a conformational change and a nucleophilic attack of Cys-35 to give the 14-membered disulfide ring in thioredoxin-S2 and the dithiol of the substrate. |
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Authors:
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G B Kallis; A Holmgren |
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Publication Detail:
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Type: Comparative Study; Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: The Journal of biological chemistry Volume: 255 ISSN: 0021-9258 ISO Abbreviation: J. Biol. Chem. Publication Date: 1980 Nov |
Date Detail:
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Created Date: 1981-01-29 Completed Date: 1981-01-29 Revised Date: 2007-11-15 |
Medline Journal Info:
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Nlm Unique ID: 2985121R Medline TA: J Biol Chem Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 10261-5 Citation Subset: IM |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acids
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analysis Bacterial Proteins / metabolism* Binding Sites Cysteine / analysis* Dithionitrobenzoic Acid / pharmacology Escherichia coli / metabolism* Hydrogen-Ion Concentration Iodoacetates Kinetics Protein Binding Thioredoxin-Disulfide Reductase / metabolism Thioredoxins / metabolism* |
| Chemical | |
Reg. No./Substance:
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0/Amino Acids; 0/Bacterial Proteins; 0/Iodoacetates; 52-90-4/Cysteine; 52500-60-4/Thioredoxins; 69-78-3/Dithionitrobenzoic Acid; EC 1.8.1.9/Thioredoxin-Disulfide Reductase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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