| Mechanism-based inactivation of dihydropyrimidine dehydrogenase by 5-ethynyluracil. | |
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MedLine Citation:
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PMID: 1544906 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Uracil analogues with appropriate substituents at the 5-position inactivated dihydropyrimidine dehydrogenase (DHPDHase). The efficiency of these inactivators was highly dependent on the size of the 5-substituent. For example, 5-ethynyluracil inactivated DHPDHase with an efficiency (kinact/Ki) that was 500-fold greater than that for 5-propynyluracil. 5-Ethynyluracil inactivated DHPDHase by initially forming a reversible complex with a Ki of 1.6 +/- 0.2 microM. This initial complex yielded inactivated enzyme with a rate constant of 20 +/- 2 min-1 (kinact). Thymine competitively decreased the apparent rate constant for inactivation of DHPDHase by 5-ethynyluracil. The absorbance spectrum of 5-ethylnyluracil-inactivated DHPDHase was different from that of reduced enzyme. These optical changes were correlated with the loss of enzymatic activity. 5-Ethynyluracil inactivated DHPDHase with a stoichiometry of 0.9 mol of inactivator per mol of active site. Enzyme inactivated with [2-14C]5-ethynyluracil retained all of the radiolabel after denaturation in 8 M urea, but lost radiolabel under acidic conditions. These results suggested that inactivation was due to covalent modification of an amino acid residue and not due to modification of a noncovalently bound prosthetic group. A radiolabeled peptide was isolated from a tryptic digest of the enzyme inactivated with [2-14C]5-ethynyluracil. The sequence of this peptide was Lys-Ala-Glu-Ala-Ser-Gly-Ala-Y-Ala-Leu-Glu-Leu-Asn-Leu-Ser-X-Pro-His-Gly- Met-Gly-Glu-Arg, where X and Y were unidentified amino acids. Since the radiolabel was lost from the peptide during the first cycle on the amino acid sequenator, the position of the radiolabeled amino acid was not determined. The amino acid residue designated by X was identified as a cysteine from previous work with DHPDHase inactivated with 5-iodouracil. In contrast to 5-ethynyluracil, 5-cyanouracil was a reversible inactivator of the enzyme. 5-Cyanouracil-inactivated enzyme slowly regained activity (t1/2 = 1.8 min) after dilution into the standard assay. DHPDHases isolated from rat, mouse, and human liver had similar sensitivities to inactivation by 5-alkynyluracils. |
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Authors:
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D J Porter; W G Chestnut; B M Merrill; T Spector |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: The Journal of biological chemistry Volume: 267 ISSN: 0021-9258 ISO Abbreviation: J. Biol. Chem. Publication Date: 1992 Mar |
Date Detail:
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Created Date: 1992-04-14 Completed Date: 1992-04-14 Revised Date: 2006-11-15 |
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: 5236-42 Citation Subset: IM |
Affiliation:
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Experimental Therapy Division, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Animals Cattle Dihydrouracil Dehydrogenase (NADP) Dithiothreitol / pharmacology Humans Kinetics Liver / enzymology* Mathematics Mice Molecular Sequence Data Oxidoreductases / antagonists & inhibitors* Peptide Fragments / isolation & purification Rats Structure-Activity Relationship Uracil / analogs & derivatives*, pharmacology* |
| Chemical | |
Reg. No./Substance:
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0/Peptide Fragments; 3483-12-3/Dithiothreitol; 59989-18-3/5-ethynyluracil; 66-22-8/Uracil; EC 1.-/Oxidoreductases; EC 1.3.1.2/Dihydrouracil Dehydrogenase (NADP) |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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