Document Detail


Mechanism-based inactivation of dihydropyrimidine dehydrogenase by 5-ethynyluracil.
MedLine Citation:
PMID:  1544906     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
D J Porter; W G Chestnut; B M Merrill; T Spector
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  267     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1992 Mar 
Date Detail:
Created Date:  1992-04-14     Completed Date:  1992-04-14     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  5236-42     Citation Subset:  IM    
Affiliation:
Experimental Therapy Division, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
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MeSH Terms
Descriptor/Qualifier:
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:
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)

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