EVIDENCE FOR OVERLAPPING ACTIVE-SITES FOR 17-ALPHA-ETHYNLESTRADIOL AND BILIRUBIN IN THE HUMAN MAJOR BILIRUBIN UDPGLUCURONOSYLTRANSFERASE

The human major bilirubin UDPglucuronosyltransferase (transferase), HU G-Brl, and its mutants were expressed in the COS-1 cells using cDNA-ba sed pSVL expression units to generate isoforms for the comparison of r elative activities with 17 alpha-ethynlestradiol (17 alpha-EE) and bil irubin, 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) Mel. Phar macol. 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 k(m) for 17 alpha-EE was 40 mu M under both pH conditions, while the V-max values were 400 and 200 pmol per hour per 300 mu g of protei n 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 k(m) values of 2.5 mu M at both pH conditions we re determined [Ritter, J. K., et al. (1993) J. Biol. Chem. 268, 23573- 23579], In this study, the generation of 17 alpha-EE and bilirubin bet a-glucuronides under both pH conditions was confirmed by the sensitivi ty of the products to beta-glucuronidase treatment, Concurrent glucuro nidation reaction mixtures containing equal amounts of wild-type and m utant proteins demonstrated the following. P270G, V273D, and five diff erent G276 mutants nearly or completely inactivated all,glucuronidatio n 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; H1 73R gave 37-60% of the normal turnover with both substrates, and V275I produced 15-24% of the normal level of glucuronide with both compound s. The most distinguishing amino acid tested was P176G which was appro ximately 50% normal for 17 alpha-EE at both pH conditions but was tota lly inactive for bilirubin, A second substitution, P285G, did not affe ct 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 ami no acids for their catalysis with the recruitment of additional amino acids to depend upon the substrate to be metabolized, Hence, amino aci d substitutions in the protein are not necessarily universally inactiv ating.