Photoaffinity Labeling of the Aglycon Binding Site of the Recombinant Human Liver UDP-Glucuronosyltransferase UGT1A6 with 7-Azido-4-methylcoumarin

doi:10.1006/abbi.1999.1300

Archives of Biochemistry and Biophysics

Volume 368, Issue 1, 1 August 1999, Pages 75-84

https://doi.org/10.1006/abbi.1999.1300 Get rights and content

Abstract

7-Azido-4-methylcoumarin (AzMC) is a fluorescent photoactive compound structurally related to 4-methylumbelliferone (4-MU), a marker substrate of the human liver recombinant UDP-glucuronosyltransferase (UGT) 1A6. AzMC was synthesized and utilized to label the substrate binding site of UGT1A6. AzMC exhibits a fluorescence spectrum with maximum excitation and emission wavelengths of 380 and 442 nm, respectively. Upon irradiation, the probe irreversibly 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 photoaffinity label. This inhibition was both time and concentration dependent and led to the calculation of an inhibition constant, k₂ = 0.113 mM min⁻¹, and dissociation constant, K_d = 2.89 mM, for the reaction. Partial photoinactivation of UGT1A6 with AzMC revealed that the probe decreased the apparent V_max of the pNP glucuronidation reaction, but not the K_m. Moreover, inhibition was partially prevented by 1-naphthol, a surrogate substrate for the enzyme, or by preincubation with an active-site directed inhibitor, 5′-O-[[(2-decanoylamino-3-phenyl-propyloxycarbonyl)amino]-sulfonyl]-2′,3′-O-isopropylideneuridine. In contrast, UDP-glucuronic acid (UDP-GlcUA) did not have any protective effect against photoinactivation and AzMC did not affect the photoaffinity labeling of UGT1A6 by 5-[β-³²P]N₃UDP-GlcUA, a photoaffinity analog of UDP-GlcUA. Additionally, in the absence of irradiation, AzMC was found to be a competitive inhibitor of 4MU glucuronidation. Collectively, these results strongly indicate that AzMC specifically binds to the UGT1A6 aglycon binding site. Amino acid alignment of phenol-binding proteins revealed a conserved motif, YXXXKXXPXP. It is possible that this motif is involved in phenol binding to UGT1A6 and other phenol-accepting proteins.

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Citation Excerpt :
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Citation Excerpt :
Several photoaffinity probes have been developed for identification of the UGT cosubstrate, UDP‐GlcUA, active site. Specifically, [β−32P]5‐azido‐UDP‐glucuronic acid ([β‐32P]5N3UDP‐GlcUA), [β‐32P]5‐azido‐UDP‐glucose ([β‐32P]5N3UDP‐Glc) (radioactive photoaffinity probes), and periodate‐oxidized [β‐32P]UDP‐GlcUA (o‐UDP‐GlcUA; affinity probe) have been designed, synthesized, characterized, and studies published (Battaglia et al., 1998; Drake et al., 1991b, 1992; Radominska and Drake, 1994; Senay et al., 1999). As far as the aglycon‐binding site(s) is concerned, three major probes are available: 7‐azido‐4‐methylcoumarin (AzMC; a fluorescent photoaffinity probe), [3H]atRA, and [3H]9cisRA (Chen and Radominska‐Pandya, 2000; Little and Radominska, 1997; Radominska‐Pandya et al., 2000, 2001).
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^☆: This work was supported in part by NIH grants to A.R.P. (DK-49715 and DK-45123) and in part by the Région Lorraine.

²: Present address: Gastroenterology Division, Brigham and Women's Hospital, 75 Francis Street, Boston MA 02115.

³: To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Slot 516, 4301 W. Markham, Little Rock, AR 72205-7199. Fax: 501-686-8169. E-mail: [email protected].

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Regular ArticlePhotoaffinity Labeling of the Aglycon Binding Site of the Recombinant Human Liver UDP-Glucuronosyltransferase UGT1A6 with 7-Azido-4-methylcoumarin☆

Abstract

Biochem. Pharmacol.

Arch. Biochem. Biophys.

FEBS Lett.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochim. Biophys. Acta

Methods Enzymol.

Methods Enzymol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Anal. Biochem.

Biochem. Pharmacol.

J. Biol. Chem.

Arch. Biochem. Biophys.

Methods Enzymol.

Life Sci.

J. Biol. Chem.

Methods Enzymol.

J. Biol. Chem.

Adv. Appl. Math.

Regular Article
Photoaffinity Labeling of the Aglycon Binding Site of the Recombinant Human Liver UDP-Glucuronosyltransferase UGT1A6 with 7-Azido-4-methylcoumarin☆