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 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.
Footnotes
This work was financially supported by the Sigrid Juselius Foundation, Finland; the Finnish Graduate School in Pharmaceutical Research; the University of Helsinki Research Foundation grant for young researchers; and the Academy of Finland [Grant 120975].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS:
- UGT
- UDP-glucuronosyltransferase
- UDPGA
- UDP-α-d-glucuronic acid
- BSA
- bovine serum albumin
- 4-MU
- 4-methylumbelliferone
- CAS
- Chemical Abstract Service
- 4-MUG
- 4-methylumbelliferone-β-d-glucuronide
- RED
- rapid equilibrium dialysis system
- HPLC
- high-performance liquid chromatography
- CI
- confidence interval
- E-UDPGA
- enzyme UDPGA.
- Received July 5, 2012.
- Accepted August 21, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|