Kinetic Characterization of the 1a Subfamily of Recombinant Human Udp-Glucuronosyltransferases

Abstract

ABSTRACT: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 firstbinding 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 M for UGT1A6 to 1256 M 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 ؊1 was calculated for the glucuronidation of scopoletin by purified UGT1A9.

Key Amino Acid Residues Responsible for the Differences in Substrate Specificity of Human UDP-Glucuronosyltransferase (UGT)1A9 and UGT1A8

“…UGT1A10 showed an 8-fold higher K m value and a 60-fold lower V max value than UGT1A10. These results suggest that UGT1A9 has the lowest apparent affinity for 4-MU among four UGT1A enzymes, consistent with previous studies (Uchaipichat et al, 2004;Luukkanen et al, 2005;Xiong et al, 2006).…”

“…Kinetic parameters by UGT1A8 could not be calculated because of the enzyme's low affinity for the substrate. These results suggest that UGT1A9 has the lowest apparent affinity for p-NP among four UGT1A enzymes, consistent with previous studies (Luukkanen et al, 2005;Xiong et al, 2006). Next, the kinetic parameters by wild-type and mutants UGT1A9 expressed in HEK293 cells were determined ( Fig.…”

Bucolome N-Glucuronide Formation: Species Differences and Identification of Human UDP-Glucuronosyltransferase Isoforms

“…They stated that the reaction involved a compulsory ordered bi bi mechanism based on analysis of inhibition profiles, in which UDP-Ga was the first binding substrate and entacapone was the second binding substrate. The mechanism of BCP-NG formation, in which the first binding substrate is UDP-Ga and the second binding substrate is BCP, agrees well with the results of Luukkanen et al [24]. The differences of kinetic parameters for BCP and UDP-Ga among animal species are believed to be due to complex reactions caused by species differences of UGT isoforms, so when drug metabolism data obtained from laboratory animals are extrapolated to human, differences in the metabolism of animal species must be taken into consideration.…”

“…Luukkanen et al [24] reported that the glucuronidation of entacapone by UGT1A9 was inhibited by 1-naphthol in an entacapone-competitive fashion and was noncompetitive with respect to UDP-Ga. Inhibition by UDP, on the other hand, was noncompetitive with respect to enta-capone and competitive with respect to UDPGA. They stated that the reaction involved a compulsory ordered bi bi mechanism based on analysis of inhibition profiles, in which UDP-Ga was the first binding substrate and entacapone was the second binding substrate.…”

Phenylalanine90 and phenylalanine93 are crucial amino acids within the estrogen binding site of the human UDP-glucuronosyltransferase 1A10

“…Microsomal protein (5 μg) from baculovirus-infected Sf9 insect cells, which were engineered to express individual UGT enzymes, was used for all the 1A family proteins. This same set of enzymes has been used in previous studies from our lab and have been show to have a high catalytic capacity for a large variety of compounds, allowing for kinetic analyses be carried out under the optimal conditions for each isoform [23,25,27,28]. UGT2B7 was over-expressed in HK293 cells.…”