KINETIC CHARACTERIZATION OF THE 1A SUBFAMILY OF RECOMBINANT HUMAN UDP-GLUCURONOSYLTRANSFERASES
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...
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Published in | Drug metabolism and disposition Vol. 33; no. 7; pp. 1017 - 1026 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
Bethesda, MD
American Society for Pharmacology and Experimental Therapeutics
01.07.2005
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Subjects | |
Online Access | Get full text |
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Summary: | 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 K m 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 k cat 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 k cat value of 1.9 s â1 was calculated for the glucuronidation of scopoletin by purified UGT1A9. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0090-9556 1521-009X |
DOI: | 10.1124/dmd.105.004093 |