Document Detail


Mutation analysis in UGT1A9 suggests a relationship between substrate and catalytic residues in UDP-glucuronosyltransferases.
MedLine Citation:
PMID:  18502788     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
UDP-glucuronosyltransferases (UGTs) catalyze the transfer of glucuronic acid from UDP-glucuronic acid to endo- and xenobiotics in our body. UGTs belong to the GT1 family of glycosyltransferases and many GT1s use a serine protease-like catalytic mechanism in which an Asp-His pair deprotonates a hydroxyl on the aglycone for nucleophilic attack on the sugar donor. The pair in human UGTs could be H37 and either D143 or D148 (UGT1A9 numbering). However, H37 is not totally conserved, being replaced by either Pro or Leu in UGT1A4 and UGT2B10. We therefore investigated the role of H37, D143 and D148 in UGT1A9 by site-directed mutagenesis, activity and kinetic measurements with several substrates. The results suggest that H37 is not critical in N-glucuronidation, but is so in O-glucuronidation. The V(max) of the H37A mutant was much less affected in N- than O-glucuronidation, while the reverse was true for the Asp mutations, particularly D143A. We suggest that this is due to the opposing properties of O- and N- nucleophiles. O-nucleophiles require the histidine to deprotonate them so that they become effective nucleophiles, while N-nucleophiles develop a formal positive charge during the reaction (RNH(2)(+)-GlcA), and thus require a negatively charged residue to stabilize the transition state.
Authors:
Anne-Sisko Patana; Mika Kurkela; Moshe Finel; Adrian Goldman
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-05-23
Journal Detail:
Title:  Protein engineering, design & selection : PEDS     Volume:  21     ISSN:  1741-0134     ISO Abbreviation:  Protein Eng. Des. Sel.     Publication Date:  2008 Sep 
Date Detail:
Created Date:  2008-08-18     Completed Date:  2008-10-03     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101186484     Medline TA:  Protein Eng Des Sel     Country:  England    
Other Details:
Languages:  eng     Pagination:  537-43     Citation Subset:  IM    
Affiliation:
Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Biocenter 3, PO Box 65, Viikinkaari 1, FIN-00014 Helsinki, Finland.
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MeSH Terms
Descriptor/Qualifier:
Amino Acids / genetics,  metabolism
Catalysis
Glucuronosyltransferase / chemistry*,  genetics
Humans
Models, Molecular*
Mutation
Substrate Specificity
Uridine Diphosphate Glucuronic Acid / metabolism*
Chemical
Reg. No./Substance:
0/Amino Acids; 2616-64-0/Uridine Diphosphate Glucuronic Acid; EC 2.4.1.17/Glucuronosyltransferase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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