Vol. 26, Issue 8, 812-817, August 1998

Interaction of Periodate-Oxidized UDP-Glucuronic Acid with Recombinant Human Liver UDP-Glucuronosyltransferase 1A6

Eric Battaglia, Nadege Terrier,¹ Magdalena Mizeracka, Claire Senay, Jacques Magdalou, Sylvie Fournel-Gigleux, and Anna Radominska-Pandya

Department of Internal Medicine, University of Arkansas for Medical Sciences (E.B., M.M., A.R.-P.), and URA CNRS 1288, Faculté de Médecine (N.T., C.S., J.M., S.F.-G.)

Sodium periodate reacts with UDP-glucuronic acid (UDP-GlcUA) to generate a reactive derivative [periodate-oxidized UDP-GlcUA (o-UDP-GlcUA)]. The ability of this analog of UDP-GlcUA to inactivate and label the human recombinant UDP-glucuronosyltransferase (UGT) UGT1A6 via the UDP-GlcUA binding site was investigated. At an o-UDP-GlcUA concentration of 20 mM, the enzymatic activity of UGT1A6 was totally inactivated after 30 min of incubation at pH 7.4. Inhibition was irreversible, time-dependent, and concentration-dependent and exhibited pseudo-first order kinetics (k_inact = 4.0 M¹·min¹). Cosubstrate protection with UDP-GlcUA was biphasic, with no protection in the first phase and almost total protection in the second phase, suggesting that at least 65% of the cross-linking occurs at the cosubstrate binding site. Partial inactivation by o-UDP-GlcUA led to a decrease in V_max, suggesting that o-UDP-GlcUA can act as an active site-directed inhibitor. Furthermore, proteins, including the UGTs, from membrane fractions of a recombinant V79 cell line expressing the UGT1A6 enzyme and from rat liver microsomes were cross-linked by in situ periodate oxidation of [-³²P]UDP-GlcUA. The present results suggest that periodate-oxidized UDP-GlcUA, which inactivates UGT1A6 by the possible formation of a Schiff base adduct with active site lysyl residues, can be used as a new affinity label for the UDP-GlcUA binding site.