RT Journal Article
SR Electronic
T1 KINETIC CHARACTERIZATION OF THE 1A SUBFAMILY OF RECOMBINANT HUMAN UDP-GLUCURONOSYLTRANSFERASES
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 1017
OP 1026
DO 10.1124/dmd.105.004093
VO 33
IS 7
A1 Leena Luukkanen
A1 Jyrki Taskinen
A1 Mika Kurkela
A1 Risto Kostiainen
A1 Jouni Hirvonen
A1 Moshe Finel
YR 2005
UL http://dmd.aspetjournals.org/content/33/7/1017.abstract
AB The initial glucuronidation rates were determined for eight recombinant human UDP-glucuronosyltransferases (UGTs) of the 1A subfamily, and the bisubstrate kinetics and inhibition patterns were analyzed. At low substrate concentrations, the reactions followed general ternary complex kinetics, whereas at higher concentrations of both substrates, the reactions were mostly characterized by ternary complex kinetics with substrate inhibition. The glucuronidation of entacapone by UGT1A9 was inhibited by 1-naphthol in a competitive fashion, with respect to entacapone, and an uncompetitive fashion, with respect to UDP-glucuronic acid (UDPGA). Its inhibition by UDP, on the other hand, was noncompetitive with respect to entacapone and competitive with respect to UDPGA. These inhibition patterns are compatible with a compulsory ordered bi bi mechanism in which UDPGA is the first-binding substrate. Despite the identical primary structure of the C-terminal halves of the UGT1A isoforms, there were marked differences in the respective Km values for UDPGA, ranging from 52 μM for UGT1A6 to 1256 μM for UGT1A8. Relative specificity constants were calculated for the eight UGT1A isoforms with 1-hydroxypyrene, 4-nitrophenol, scopoletin, 4-methylumbelliferone, and entacapone as aglycone substrates. The results demonstrated that seven of the UGT1A isoforms are capable of conjugating phenolic substrates with similar highest kcat values, and UGT1A4 has a lower relative turnover rate. The highest specificity constants were obtained for 1-hydroxypyrene, even with UGT1A6, which has been regarded as a specific isoform for small planar phenols. A kcat value of 1.9 s–1 was calculated for the glucuronidation of scopoletin by purified UGT1A9. The American Society for Pharmacology and Experimental Therapeutics