title: Mutation analysis in UGT1A9 suggests a relationship between substrate and catalytic residues in UDP-glucuronosyltransferases.
abstract: 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.affiliation: Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Biocenter 3, PO Box 65, Viikinkaari 1, FIN-00014 Helsinki, Finland.
authors.forename: Anne-Sisko
authors.initials: AS
authors.lastname: Patana
authors.forename: Mika
authors.initials: M
authors.lastname: Kurkela
authors.forename: Moshe
authors.initials: M
authors.lastname: Finel
authors.forename: Adrian
authors.initials: A
authors.lastname: Goldman
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D013379:Substrate Specificity; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D014535:Uridine Diphosphate Glucuronic Acid; D014453:Glucuronosyltransferase