Received for publication June 8, 2005.
Revised August 26, 2005.
Accepted for publication August 29, 2005.

Glycosidation of an Endothelin ET_A Receptor Antagonist and Diclofenac in Human Liver Microsomes: Aglycone-dependent UDP-sugar Selectivity

Abstract

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 ET_A receptor antagonist Compound A to form an acyl glucuronide 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 appeared 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 µM. 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 µM and 1277 ± 149 µM, > 10-folds 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 towards 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 mechanism may involve enzyme conformational changes associated with Compound A binding to the enzyme.

Key words: glucuronidation, liver microsomes, UDP glucuronyltransferases