Opioids Bind to the Amino Acids 84 to 118 of UDP-Glucuronosyltransferase UGT2B7

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

doi:10.1124/mol.63.2.283

Cited by 28 publications

(22 citation statements)

References 12 publications

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“…Studies with chimeric and fusion proteins are generally consistent with the hypothesis that aglycone selection is determined by the amino terminal portion of UGT2B subfamily enzymes (Mackenzie, 1990;Ritter et al, 1992;Li et al, 1997;Lewis et al, 2007). Site-directed mutagenesis and NMR spectroscopy have further implicated a limited number of amino acids in the N-terminal region of UGT 2B7, 2B15, and 2B17 in substrate binding (Dubois et al, 1999;Coffman et al, 2003). However, the existence of multiple substrate binding domains within the UGT2B7 active site has not been explored in a systematic manner.…”

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confidence: 49%

Kinetic Modeling of the Interactions between 4-Methylumbelliferone, 1-Naphthol, and Zidovudine Glucuronidation by UDP-Glucuronosyltransferase 2B7 (UGT2B7) Provides Evidence for Multiple Substrate Binding and Effector Sites

Uchaipichat¹,

Galetin²,

Houston³

et al. 2008

Mol Pharmacol

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Interactions between the UGT2B7-catalyzed glucuronidation of zidovudine (AZT), 4-methylumbelliferone (4MU), and 1-naphthol (1NP) were analyzed using multisite and empirical kinetic models to explore the existence of multiple substrate and effector binding sites within this important drug metabolizing enzyme. 4MU and 1NP glucuronidation by UGT2B7 exhibit sigmoidal kinetics characteristic of homotropic cooperativity (autoactivation), which may be modeled assuming the existence of two equivalent, interacting substrate binding sites. In contrast, UGT2B7-catalyzed AZT glucuronidation follows hyperbolic (Michaelis-Menten) kinetics. Although 4MU and 1NP decreased the binding affinity of AZT, the kinetics of AZT glucuronidation changed from hyperbolic to sigmoidal in the presence of both modifiers. Data were well described by a generic two-substrate binding site model in which there is no interaction between the sites in the absence of 4MU or 1NP, but heterotropic cooperativity results from the binding of modifier. Inhibition of 4MU and 1NP glucuronidation by AZT and interactions between 4MU and 1NP required more complex three-site models, where the modifier acts via a distinct effector site to alter either substrate binding affinity or V max without affecting the homotropic cooperativity characteristic of 4MU and 1NP glucuronidation. It is noteworthy that 1NP inhibited 4MU glucuronidation, whereas 4MU activated 1NP glucuronidation. The results are consistent with the existence of two "catalytic" sites for each substrate within the UGT2B7 active site, along with multiple effector sites. The multiplicity of binding and effector sites results in complex kinetic interactions between UGT2B7 substrates, which potentially complicates inhibition screening studies.Conjugation with glucuronic acid ("glucuronidation") is responsible for the elimination and detoxification of a structurally diverse range of compounds that includes drugs, dietary chemicals, environmental pollutants, and endogenous compounds (Miners and Mackenzie, 1991;Radominska-Pandya et al., 1999;Tukey and Strassburg, 2000). Glucuronidation reactions are catalyzed by the enzyme UDP-glucuronosyltransferase (UGT). Consistent with its broad substrate profile, UGT comprises a "superfamily" of enzymes that has been further classified into two families, UGT1 and UGT2, based on evolutionary divergence (Mackenzie et al., 2005). Of the seventeen human UGT proteins characterized to date, UGT2B7 is arguably the most important in terms of drug metabolism. UGT2B7 catalyzes the glucuronidation of many opioids and nonsteroidal anti-inflammatory drugs, as well as some anticonvulsants, epirubicin, and zidovudine (Jin et al

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mentioning

confidence: 49%

Kinetic Modeling of the Interactions between 4-Methylumbelliferone, 1-Naphthol, and Zidovudine Glucuronidation by UDP-Glucuronosyltransferase 2B7 (UGT2B7) Provides Evidence for Multiple Substrate Binding and Effector Sites

Uchaipichat¹,

Galetin²,

Houston³

et al. 2008

Mol Pharmacol

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“…Previous studies (e.g., Dubois et al, 1999;Coffman et al, 2003;Xiong et al, 2006;Lewis et al, 2007) have generally linked the aglycone substrate selectivity of individual UGT enzymes with residues or domains spanning positions 60 to 194. The present study demonstrates that residues 36 and 40 of UGT1A3 and UGT1A4, which are close to the amino terminus of the mature UGT protein, are pivotal for the respective selectivities of these enzymes toward planar phenols and tertiary amines.…”

Section: Discussionmentioning

confidence: 99%

Critical Roles of Residues 36 and 40 in the Phenol and Tertiary Amine Aglycone Substrate Selectivities of UDP-Glucuronosyltransferases 1A3 and 1A4

Kubota¹,

Lewis²,

Elliot³

et al. 2007

Mol Pharmacol

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Despite high sequence identity, UGT1A3 and UGT1A4 differ in terms of substrate selectivity. UGT1A3 glucuronidates the planar phenols 1-naphthol (1-NP) and 4-methylumbelliferone (4-MU), whereas UGT1A4 converts the tertiary amines lamotrigine (LTG) and trifluoperazine (TFP) to quaternary ammonium glucuronides. Residues 45 to 154 (which incorporate 21 of the 35 amino acid differences) and 45 to 535 were exchanged between UGT1A3 and UGT1A4 to generate UGT1A3-4 (45-535) , UGT1A3-4 (45-154) -3, , and UGT1A4-3 (45-154) -4 hybrid proteins. Although differences in kinetic parameters were observed between the parent enzymes and chimeras, and UGT1A4-3 (45-154) -4 [but not and -3] retained the capacity to glucuronidate LTG and TFP. Likewise, and -3 retained the capacity to glucuronidate 1-NP and 4-MU, but UGT1A4-3 (45-535) and UGT1A4-3 (45-154) -4 exhibited low or absent activity. Within the first 44 residues, UGT1A3 and UGT1A4 differ in sequence at positions 36 and 40. "Reciprocal" mutagenesis was performed to generate the UGT1A3(I36T), UGT1A3(H40P), UGT1A4(T36I), and UGT1A4 (P40H) mutants. The T36I and P40H mutations in UGT1A4 reduced in vitro clearances for LTG and TFP glucuronidation by Ͼ90%. Conversely, the I36T and H40P mutations in UGT1A3 reduced the in vitro clearances for 1-NP and 4-MU glucuronidation by Ͼ90%. Introduction of the single H40P mutation in UGT1A3 conferred LTG and TFP glucuronidation, whereas the single T36I mutation in UGT1A4 conferred 1-NP and 4-MU glucuronidation. Thus, residues 36 and 40 of UGT1A3 and UGT1A4 are pivotal for the respective selectivities of these enzymes toward planar phenols and tertiary amines, although other regions of the proteins influence binding affinity and/or turnover.

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“…With respect to morphine, hepatic glucuronidation leading to the formation of morphine-6-glucuronide and morphine-3-glucuronide is the most important pathway of opioid metabolism (for review see: Tegeder et al, 1999). UDP-glucuronosyl transferase 2B7 (UGT2B7) is the relevant isozyme (Coffman et al, 2003;Thorn et al, 2009) and its activity as well as UGT2B7 promotor variants influence the morphine metabolite/morphine ratio following oral morphine administration (Darbari et al, 2008;Tateishi et al, 2003). Plasma concentrations of morphine, morphine-6-glucuronide and morphine-3-glucuronide vary widely, both in healthy subjects and in patients suffering from severe pain (e.g.…”

Section: Introductionmentioning

confidence: 99%