INTERACTION OF PERIODATE-OXIDIZED UDP-GLUCURONIC ACID WITH RECOMBINANT HUMAN LIVER UDP-GLUCURONOSYLTRANSFERASE 1A6

Sodium periodate reacts with UDP-glucuronic acid (UDP-GlcUA) to genera te a reactive derivative [periodate-oxidized UDP-GlcUA (o-UDP-GlcUA)]. The ability of this analog of UDP-GlcUA to inactivate and label the h uman recombinant UDP-glucuronosyltransferase (UGT) UGT1A6 via the UDP- GlcUA binding site was investigated. At an o-UDP-GlcUA concentration o f 20 mM, the enzymatic activity of UGT1A6 was totally inactivated afte r 30 min of incubation at pH 7.4. Inhibition was irreversible, time-de pendent, and concentration-dependent and exhibited pseudo-first order kinetics (k(inact) = 4.0 M-1.min(-1)). 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 inac tivation 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, p roteins, including the UGTs, from membrane fractions of a recombinant V79 cell line expressing the UGT1A6 enzyme and from rat liver microsom es were cross-linked by in situ periodate oxidation of [beta-P-32]UDP- GlcUA. The present results suggest that periodate-oxidized UDP-GlcUA, which inactivates UGT1A6 by the possible formation of a Schiff base ad duct with active site lysyl residues, can be used as a new affinity la bel for the UDP-GlcUA binding site.