Interaction of periodate-oxidized UDP-glucuronic acid with recombinant human liver UDP-glucuronosyltransferase 1A6.
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 (kinact = 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 inactivation by o-UDP-GlcUA led to a decrease in Vmax, 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 [beta-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.
Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
E
E
Battaglia
N
N
Terrier
M
M
Mizeracka
C
C
Senay
J
J
Magdalou
S
S
Fournel-Gigleux
A
A
Radominska-Pandya
D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D014453:Glucuronosyltransferase / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D010504:Periodic Acid / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010761:Phosphorus Radioisotopes; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D010504:Periodic Acid; D014535:Uridine Diphosphate Glucuronic Acid; C110655:UDP-glucuronosyltransferase, UGT1A6; D014453:Glucuronosyltransferase