RESUMO
The human UDP-glucuronosyltransferase 1A6 (UGT1A6) isoform is actively involved in the detoxication of phenolic compounds. In an effort to gain insight on active-site amino acids, we investigated the functional relevance of cysteinyl residues in the glucuronidation process. The enzyme was irreversibly inactivated upon exposure to thiol-specific reagents, especially N-phenylmaleimide. Site-directed mutagenesis of the conserved Cys126 into valine led to a fully inactive mutant, whereas conservative substitution with serine significantly restored the glucuronidation activity toward 4-methylumbelliferone used as a reference substrate. This mutant exhibited a reduced affinity toward the acceptor substrate, as evidenced by a 10-times increase in K(m) value, compared to the wild-type enzyme. The two mutations did not alter the stability of UGT1A6 nor change the subcellular localization of the protein in the endoplasmic reticulum of recombinant cells. These results support the conclusion that Cys126 is an essential residue for the integrity of the substrate binding site of UGT1A6.
