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.
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 compulsory-order ternary-complex, with UDP-α-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.
Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland.
Nenad
N
Manevski
Jari
J
Yli-Kauhaluoma
Moshe
M
Finel
D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007313:Insecta; D007700:Kinetics; D009284:Naphthols / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D000090264:UDP-Glucuronosyltransferase 1A9; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism*
D009284:Naphthols; C000715591:UGT1A9 protein, human; D014535:Uridine Diphosphate Glucuronic Acid; D012710:Serum Albumin, Bovine; C029350:1-naphthol; D006923:Hymecromone; D014453:Glucuronosyltransferase; D000090264:UDP-Glucuronosyltransferase 1A9