title: The human UDP-glucuronosyltransferase: identification of key residues within the nucleotide-sugar binding site.
abstract: UDP-glucuronosyltransferases (UGTs) play important roles in the metabolism, detoxification,and clearance of many different xenobiotics, including drugs and endogenous compounds. Structural information about these membrane-bound enzymes of the endoplasmic reticulum is limited. We do not know the identity or the location of the key residues for catalysis and binding of the aglycone substrate and the cosubstrate UDP-glucuronic acid (UDPGA). One suggestion was that His371 (UGT1A6 numbering) is the "catalytic base" that deprotonates the phenol group. We have now re-examined this hypothesis by analyzing the activities of the corresponding mutants, 6H371A (in UGT1A6) and the 9H369A (in UGT1A9). The K(m) values of mutant 6H371A for scopoletin and UDPGA were higher by 4- and 11-fold, respectively, than in UGT1A6. The K(d) for the enzyme-UDPGA complex, derived from bisubstrate kinetics, was about 9-fold higher in 6H371A than in UGT1A6, indicating severely impaired cosubstrate binding by the mutant. The effect of mutation on V(max) was large in UGT1A6 but variable in UGT1A9, suggesting that His371 does not play the catalytic role previously hypothesized. In both UGTs, the E379A mutation (UGT1A6 numbering) had an effect similar to that of the H371A mutations. A homology model of the putative UDPGA binding region of UGT1A6 was built using distant homologous protein structures from the "GT1 class." The combined results of activity determinations, kinetic analyses, and modeling strongly suggest that His371 and Glu379 are directly involved in UDPGA binding but are not the general acid or general base.
authors.affiliation: Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Finland.
authors.forename: Anne-Sisko
authors.initials: AS
authors.lastname: Patana
authors.forename: Mika
authors.initials: M
authors.lastname: Kurkela
authors.forename: Adrian
authors.initials: A
authors.lastname: Goldman
authors.forename: Moshe
authors.initials: M
authors.lastname: Finel
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D012603:Scopoletin / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D020719:Glucuronides; D009711:Nucleotides; D014453:Glucuronosyltransferase; D012603:Scopoletin