A NOVEL UDP-GLC-SPECIFIC GLUCOSYLTRANSFERASE CATALYZING THE BIOSYNTHESIS OF 6-O-GLUCOSIDES OF BILE-ACIDS IN HUMAN LIVER-MICROSOMES

Two active site-directed photoaffinity analogs, 5-[beta-P-32]azido-UDP-glucuronic acid and 5-[beta-P-32]azido-UDP-glucose, were used for the characterization of UDP-sugar-utilizing enzymes in human liver microsomes. Both compounds were recognized by human microsomal proteins: major photolabeled bands of 50-56 kDa were detected. Both photoincorporations were competitively decreased by increasing concentrations of either UDP-Glc or UDP-GlcUA, indicating a high affinity for both nucleotides. The patterns of photoaffinity labeling in the 50-56-kDa range by the two probes were significantly different, indicating the presence of different UDP-GlcUA- and UDP-Glc-specific enzymes of similar molecular mass. The presence of a UDP-Glc-dependent transferase was confirmed by the identification of an enzymatic activity catalyzing the formation of glucosides of the 6alpha-hydroxylated bile acid hyodeoxycholic acid (3alpha,6alpha-diOH (HDCA)) in the presence of UDP-Glc. The specific activity of 1.5-3.2 nmol/min/mg of protein was similar to that of 6alpha-glucuronidation of HDCA. The apparent K(m) for UDP-Glc estimated with HDCA was 280 muM, and the formation of HDCA glucosides was strongly inhibited by UDP-GlcUA (apparent K(i) = 7 muM). Evidence is presented that HDCA-specific UDP-glucuronosyltransferase clone UGT2B4) expressed in V79 cells is not involved in glucosidation of HDCA and is not photolabeled with 5-[beta-P-32]azido-UDP-Glc. Rigorous structure identification of the biosynthetic product proved that HDCA was glucosidated at the 6-position. Thus, this UDP-Glc-dependent activity catalyzing the biosynthesis of 6-O-glucosides of 6alpha-hydroxylated bile acids represents a new pathway in the metabolism of these bile acids.