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 bilirubin and phenol UDP-glucuronosyltransferases (transferase) has been critical to the determination of genetic defects in Crigler-Najjar Type I patients. The complex (UGT1A-UGT1G) codes for at least two bilirubin, three bilirubin-like, and two phenol transferases. Seven different exons 1, each with an upstream promoter and each encoding the amino terminus of an isoform, are arrayed in series with four common exons (encoding seven identical carboxyl termini) in the 3′-region of the locus. Predictably, a critical mutation in a common exon inactivates the entire locus. A deleterious mutation in an exon 1, as we report here for the UGT1A gene in a Crigler-Najjar Type I patient, predictably affects the amino terminus of that single isoform. The code for the predominant bilirubin isozyme, the HUG-Br1 protein, is missing the phenylalanine codon at position 170 in exon 1 of UGT1A, abolishing a conserved diphenylalanine. We demonstrate that, at the pH (7.6) routinely used for bilirubin glucuronidation studies, both the HUG-Br1 protein and human liver microsomes have approximately one-third the activity seen at the major pH optimum of 6.4 and at low ionic strength. The altered isozyme with nearly normal activity at pH 7.6 is inactive at pH 6.4, a result consistent with the definition of a pH-sensitive mutant. The Km value for bilirubin using the wild-type protein is ∼2.5 μM at both pH 6.4 and 7.6 and that for the mutant is 5.0 μM 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 critical for metabolizing the lipophile-like substrate. The low ion/pH requirements for bilirubin glucuronidation may signal the basis for the distribution of these isozymes to an organelle (endoplasmic reticulum) that can establish compatible conditions/compartments for each catalysis.
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Nov 5 1993|
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