Opioids bind to the amino acids 84 to 118 of UDP-glucuronosyltransferase UGT2B7

Birgit L Coffman; William R Kearney; Shawn Goldsmith; Boyd M Knosp; Thomas R Tephly

doi:10.1124/mol.63.2.283

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Opioids bind to the amino acids 84 to 118 of UDP-glucuronosyltransferase UGT2B7

Journal article

Peer reviewed

Opioids bind to the amino acids 84 to 118 of UDP-glucuronosyltransferase UGT2B7

Birgit L Coffman, William R Kearney, Shawn Goldsmith, Boyd M Knosp and Thomas R Tephly

Molecular pharmacology, Vol.63(2), pp.283-288

02/2003

DOI: 10.1124/mol.63.2.283

PMID: 12527799

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Abstract

The UDP-glucuronosyltransferase UGT2B7 is an important human UGT isoform that catalyzes the conjugation of many endogenous and exogenous compounds, among them opioids, resulting in the formation of D-glucuronides. The binding site of the aglycone is located in the N-terminal half of the protein. Using NMR analysis, we demonstrate that the opioid binding site in UGT2B7 is within the 84 to 118 N-terminal amino acids. Three maltose binding protein-UGT2B7 fusion proteins, 2B7F3 and 2B7F4 incorporating the amino acids 24 to 118 and 24 to 96 of UGT2B7, respectively, and 2B7F5 incorporating amino acids 84 to 118 of UGT2B7 were expressed in Escherichia coli and purified by affinity chromatography. NMR analysis showed that morphine was bound to the fusion protein 2B7F3 with a K(D) value similar to the K(D) values obtained for the previously produced fusion proteins, which included amino acids 24 to 180. Morphine did not bind to 2B7F4, but it did bind to 2B7F5. Both NMR 1-D spectra and NOESY experiments indicated that the 2B7F5 protein was mediating magnetization transfer within the morphine. These results allowed us to predict and model a binding site within the amino acids 96 to 101 of UGT2B7. A mutant fusion protein 2B7F3 with the substitution D99A was produced, and the NMR spectroscopy analysis of the protein supported the model. A marked reduction of morphine binding was observed when the charged aspartate was substituted with alanine.

Amino Acid Sequence

Animals

Aspartic Acid - genetics

Binding Sites

Carrier Proteins - biosynthesis

Carrier Proteins - genetics

Glucuronosyltransferase - chemistry

Glucuronosyltransferase - genetics

Glucuronosyltransferase - metabolism

Humans

Magnetic Resonance Spectroscopy

Maltose-Binding Proteins

Models, Molecular

Molecular Sequence Data

Morphine - pharmacology

Mutagenesis, Site-Directed

Protein Conformation

Recombinant Fusion Proteins - biosynthesis

Details

Title: Subtitle: Opioids bind to the amino acids 84 to 118 of UDP-glucuronosyltransferase UGT2B7
Creators: Birgit L Coffman - University of Iowa
William R Kearney
Shawn Goldsmith
Boyd M Knosp
Thomas R Tephly
Resource Type: Journal article
Publication Details: Molecular pharmacology, Vol.63(2), pp.283-288
DOI: 10.1124/mol.63.2.283
PMID: 12527799
ISSN: 0026-895X
eISSN: 1521-0111
Grant note: GM26221 / NIGMS NIH HHS
Language: English
Date published: 02/2003
Academic Unit: Radiology; Neuroscience and Pharmacology; Medicine Administration
Record Identifier: 9984383914902771

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