Evidence for overlapping active sites for 17 alpha-ethynlestradiol and bilirubin in the human major bilirubin UDPglucuronosyltransferase.
The human major bilirubin UDP glucuronosyltransferase (transferase), HUG-Brl, and its mutants were expressed in the COS-1 cells using cDNA-based pSVL expression units to generate isoforms for the comparison of relative activities with 17 alpha-ethynlestradiol (17 alpha-EE) and bilirubin, its natural substrate. In comparison to bilirubin, 17 alpha-EE was a good substrate for HUG-Br1 under typical assay conditions of pH 7.2, confirming published studies [Ebner, T., et al. (1993) Mol. Pharmacol. 43, 649-654]. It was further shown that the estrogen derivative is 1.2-2-fold more effective as a substrate at pH 6.4 than at pH 7.2. The km for 17 alpha-EE was 40 microM under both pH conditions, while the Vmax values were 400 and 200 pmol per hour per 300 micrograms of protein at pH 6.4 and 7.2, respectively. The pattern of glucuronidation was similar for both bilirubin and 17 alpha-EE. Previously, a ratio of 2-3-fold more activity for bilirubin glucuronidation at pH 6.4 versus 7.6 was established, and km values of 2.5 microM at both pH conditions were determined [Ritter, J.K., et al. (1993) J. Biol. Chem. 268, 23573-23579]. In this study, the generation of 17 alpha-EE and bilirubin beta-glucuronides under both pH conditions was confirmed by the sensitivity of the products to beta-glucuronidase treatment. Concurrent glucuronidation reaction mixtures containing equal amounts of wild-type and mutant proteins demonstrated the following. P270G, V273D, and five different G276 mutants nearly or completely inactivated all glucuronidation at both pH levels. V273Q generated 81-94% of the normal activity for 17 alpha-EE and 42% of the normal activity for bilirubin turnover; H173R gave 37-60% of the normal turnover with both substrates, and V275I produced 15-24% of the normal level of glucuronide with both compounds. The most distinguishing amino acid tested was P176G which was approximately 50% normal for 17 alpha-EE at both pH conditions but was totally inactive for bilirubin. A second substitution, P285G, did not affect 17 alpha-EE turnover but was 50% normal for bilirubin. The parallel effects on the metabolism of both substrates by some mutants and the opposite results from two mutants are evidence for a common set of amino acids for their catalysis with the recruitment of additional amino acids to depend upon the substrate to be metabolized. Hence, amino acid substitutions in the protein are not necessarily universally inactivating.
Ciotti
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Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1830, USA.
Owens
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001663:Bilirubin / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004997:Ethinyl Estradiol / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; C019787:bilirubin glucuronoside glucuronosyltransferase; D004997:Ethinyl Estradiol; D014453:Glucuronosyltransferase; D001663:Bilirubin