GLYCOSIDATION OF AN ENDOTHELIN ETA RECEPTOR ANTAGONIST AND DICLOFENAC IN HUMAN LIVER MICROSOMES: AGLYCONE-DEPENDENT UDP-SUGAR SELECTIVITY□S

Following the finding that UGT2B7 catalyzes the transfer of the glycosyl group from both UDP-glucuronic acid (UDP-GlcA) and UDP-glucose (UDP-Glc) to an endothelin ETA receptor antagonist, Compound A [()-(5S,6R,7R)-2-isopropylamino-7-[4-methoxy-2-((2R)-3-methoxy-2-methylpropyl)-5-(3,4-methylenedioxyphenyl)cy-clopenteno[1,2-b] pyridine 6-carboxylic acid], to form an acyl gluc-uronide and a glucoside (Tang et al., 2003), two additional nucleotide sugars [UDP-galactose (UDP-gal) and UDP-N-acetyl glucosamine (UDP-GlcNAc)] were examined as cosubstrates in human liver microsomes. It was found that UDP-gal, but not UDP-GlcNAc, also served as a sugar donor primarily through catalysis by UGT2B7, although at a significantly reduced catalytic rate. These three UDP-sugars showed pH-dependent kinetics and ap-peared to compete with each other, with IC50 values parallel to their respective apparent Km values. In contrast, only UDP-GlcA served as the sugar donor for the conjugation of diclofenac, a known UGT2B7 substrate, with an apparent Km for UDP-GlcA of 96 17 M. UDP-Glc and UDP-gal, two futile sugar donors for diclofenac, were found to competitively inhibit the glucuronidation of this aglycone. Different from the case with Compound A, UDP-Glc and UDP-gal displayed Ki values of 2054 108 M and 1277 149 M,>10-fold greater than the Km for UDP-GlcA, indicating that these two nucleotide sugars were also capable of binding to the enzyme but with a lower affinity. The findings of this study indicate that the selectivity of UGT2B7 toward UDP-sugars is aglycone-dependent. With Compound A as the acceptor substrate, human UGT2B7 becomes more accommodative in the transfer of the glycosyl group from UDP-sugars beyond UDP-GlcA. The mecha-nism may involve enzyme conformational changes associated with Compound A binding to the enzyme. The UDP-glycosyltransferases are members of a superfamily of enzymes that catalyze the covalent conjugation of a variety of agly-cone substrates (acceptors) with the glycosyl moiety of UDP-sugars (donors) to form a glycosidic bond. The UDP-sugar donor may b