Photoaffinity labeling of the aglycon binding site of the recombinant human liver UDP-glucuronosyltransferase UGT1A6 with 7-azido-4-methylcoumarin

Citation
C. Senay et al., Photoaffinity labeling of the aglycon binding site of the recombinant human liver UDP-glucuronosyltransferase UGT1A6 with 7-azido-4-methylcoumarin, ARCH BIOCH, 368(1), 1999, pp. 75-84
Citations number
39
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
ISSN journal
00039861 → ACNP
Volume
368
Issue
1
Year of publication
1999
Pages
75 - 84
Database
ISI
SICI code
0003-9861(19990801)368:1<75:PLOTAB>2.0.ZU;2-4
Abstract
7-Azido-4-methylcoumarin (AzMC) is a fluorescent photoactive compound struc turally related to 4-methylumbelliferone (4-MU), a marker substrate of the human liver recombinant UDP-glucuronosyltransferase (UGT) 1A6, AzMC was syn thesized and utilized to label the substrate binding site of UGT1A6. AzMC e xhibits a fluorescence spectrum with maximum excitation and emission wavele ngths of 380 and 442 nm, respectively. Upon irradiation, the probe irrevers ibly inhibited glucuronidation activity measured with para-nitrophenol (pNP ) as substrate and interacted with UGT1A6 according to a saturable process indicative of reversible binding before covalent incorporation of the photo affinity label. This inhibition was both time and concentration dependent a nd led to the calculation of an inhibition constant, k(2) = 0.113 mM min(-1 ), and dissociation constant, K-d = 2.89 mM, for the reaction. Partial phot oinactivation of UGT1A6 with AzMC revealed that the probe decreased the app arent V-max of the PNP glucuronidation reaction, but not the K-m. Moreover, inhibition was partially prevented by 1-naphthol, a surrogate substrate fo r the enzyme, or by preincubation with an active-site directed inhibitor, 5 '-O-[[(2-decanoylamino-3-phenyl-propyloxycarbonyl)amino]-sulfonyl]-2',3'-O- isopropylideneuridine. Ln contrast, UDP-glucuronic acid (UDP-GlcUA) did not have any protective effect against photoinactivation and AzMC did not affe ct the photoaffinity labeling of UGT1A6 by 5-[beta-P-32]N3UDP-GlcUA, a phot oaffinity analog of UDP-GlcUA, Additionally, in the absence of irradiation, AzMC was found to be a competitive inhibitor of 4MU glucuronidation. Colle ctively, these results strongly indicate that AzMC specifically binds to th e UGT1A6 aglycon binding site. Amino acid alignment of phenol-binding prote ins revealed a conserved motif, YXXXKXXPXP, It is possible that this motif is involved in phenol binding to UGT1A6 and other phenol-accepting proteins . (C) 1999 Academic Press.