A PHENYLALANINE CODON DELETION AT THE UGT1 GENE-COMPLEX LOCUS OF A CRIGLER-NAJJAR TYPE-I PATIENT GENERATES A PH-SENSITIVE BILIRUBIN UDP-GLUCURONOSYLTRANSFERASE

The characterization (Ritter, J. K., Chen, F., Sheen, Y. Y., Tran, H. M., Kimura, S., Yeatman, M. T., and Owens, I. S. (1992) J. Biol. Chem. 267,3257-3261) of the single-copy UGT1 gene complex encoding both bil irubin and phenol UDP-glucuronosyltransferases (transferase) has been critical to the determination of genetic defects in Crigler-Najjar Typ e I patients. The complex (UGT1A-UGT1G) codes for at least two bilirub in, three bilirubin-like, and two phenol transferases. Seven different exons 1, each with an upstream promoter and each encoding the amino t erminus of an isoform, are arrayed in series with four common exons (e ncoding seven identical carboxyl termini) in the 3'-region of the locu s. Predictably, a critical mutation in a common exon inactivates the e ntire locus. A deleterious mutation in an exon 1, as we report here fo r the UGT1A gene in a Crigler-Najjar Type I patient, predictably affec ts the amino terminus of that single isoform. The code for the predomi nant bilirubin isozyme, the HUG-Br1 protein, is missing the phenylalan ine codon at position 170 in exon 1 of UGT1A, abolishing a conserved d iphenylalanine. We demonstrate that, at the pH (7.6) routinely used fo r bilirubin glucuronidation studies, both the HUG-Br1 protein and huma n liver microsomes have approximately one-third the activity seen at t he major pH optimum of 6.4 and at low ionic strength. The altered isoz yme with nearly normal activity at pH 7.6 is inactive at pH 6.4, a res ult consistent with the definition of a pH-sensitive mutant. The K(m) value for bilirubin using the wild-type protein is approximately 2.5 m uM at both pH 6.4 and 7.6 and that for the mutant is 5.0 muM at pH 7.6 . The structure of the wild-type enzyme compared to that of the mutant indicates that hydrophobic properties at the active center are critic al for metabolizing the lipophile-like substrate. The low ion/pH requi rements for bilirubin glucuronidation may signal the basis for the dis tribution of these isozymes to an organelle (endoplasmic reticulum) th at can establish compatible conditions/compartments for each catalysis .