UDP-Glucuronic Acid Binds First and the Aglycone Substrate Binds Second to Form a Ternary Complex in UGT1A9-Catalyzed Reactions, in Both the Presence and Absence of Bovine Serum Albumin

Abstract

ABSTRACT:The presence of bovine serum albumin (BSA) largely modulates the enzyme kinetics parameters of the human UDP-glucuronosyltransferase (UGT) 1A9, increasing both the apparent aglycone substrate affinity of the enzyme and its limiting reaction velocity (Drug Metab Dispos 39:2117-2129, 2011). For a better understanding of the BSA effects and an examination of whether its presence changes the catalytic mechanism, we have studied the enzyme kinetics of 4-methylumbelliferone glucuronidation by UGT1A9 in the presence and absence of 0.1% BSA, using bisubstrate enzyme kinetic experiments, in both the forward and reverse directions, as well as product and dead-end inhibition. The combined results strongly suggest that the reaction mechanism of UGT1A9, and presumably other human UGTs as well, involves the formation of a compulsoryorder ternary-complex, with UDP-␣-D-glucuronic acid (UDPGA) as the first binding substrate. Based on the enzyme kinetic parameters measured for the forward and reverse reactions, the equilibrium constant of the overall reaction was calculated (Keq ‫؍‬ 574) and the relative magnitudes of the reaction rate constants were elucidated. The inclusion of BSA in the bisubstrate kinetic experiments quantitatively changed the apparent enzyme kinetic parameters, presumably by removing internal inhibitors that bind to the binary enzyme-UDPGA (E-UDPGA) complex, as well as to the ternary E-UDPGA-aglycone complex. Nevertheless, the underlying compulsory-order ternary-complex mechanism with UDPGA binding first is the same in both the absence and presence of BSA. The results offer a novel understanding of UGT enzyme kinetic mechanism and BSA effects.

Application of a Physiologically Based Pharmacokinetic Model to Assess Propofol Hepatic and Renal Glucuronidation in Isolation: Utility of In Vitro and In Vivo Data

“…Similar effects were observed at both 1 and 2% BSA, suggesting that the effect could not be simply rationalized by the differential concentration ratio of microsomal protein to BSA. Current findings are in contrast to an increase in V max reported for other UGT1A9 substrates in the presence of BSA and at low microsomal protein concentrations (Manevski et al, 2011(Manevski et al, , 2012. However, differences in assay conditions in the published studies prevent direct comparison with the findings herein; further investigation is required to confirm whether these effects on V max are directly associated with the use of BSA at very low protein concentrations.…”

Reaction Phenotyping: Advances in the Experimental Strategies Used to Characterize the Contribution of Drug-Metabolizing Enzymes

“…The reasoning was akin to that described for CYP2C9, whereby BSA was acting as a sink to bind free fatty acids released from the microsomal membranes during preparation, thus avoiding the competitive inhibition imparted by the fatty acids. Additional UGT/BSA studies conducted in a separate laboratory confirmed the effects of BSA on UGT2B7, and provided evidence that not only was the K m decreased, but the V max of UGT1A9 was also increased (Manevski et al, 2011(Manevski et al, , 2012.…”

Challenges and Opportunities with Non-CYP Enzymes Aldehyde Oxidase, Carboxylesterase, and UDP-Glucuronosyltransferase: Focus on Reaction Phenotyping and Prediction of Human Clearance

“…This has been shown to work only for UGTs 1A1, 1A4 in microsomes but the data was not corroborated in Supersomes. It has also been documented for UGT1A9 in sf9 cell lysate supernatants but not validated in Supersomes or microsomes (101, 102). Moreover, there is no evidence yet that this is a valid method of inhibiting hepatic and other extra-hepatic UGTs either in microsomes or recombinant systems.…”