title: 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.
abstract: 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 Vmax 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.
authors.affiliation: Department of Clinical Pharmacology, Flinders University, Flinders Medical Centre, Adelaide, Australia.
authors.forename: Verawan
authors.initials: V
authors.lastname: Uchaipichat
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Galetin
authors.forename: J Brian
authors.initials: JB
authors.lastname: Houston
authors.forename: Peter I
authors.initials: PI
authors.lastname: Mackenzie
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Williams
authors.forename: John O
authors.initials: JO
authors.lastname: Miners
mesh_terms: D000963:Antimetabolites / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D020719:Glucuronides / Q000378:metabolism / Q000494:pharmacology; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D007202:Indicators and Reagents / Q000378:metabolism / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D009284:Naphthols / Q000378:metabolism* / Q000494:pharmacology; D013379:Substrate Specificity; D014162:Transfection; D015215:Zidovudine / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000963:Antimetabolites; D020719:Glucuronides; D007202:Indicators and Reagents; D009284:Naphthols; C029350:1-naphthol; D006923:Hymecromone; D015215:Zidovudine; C491474:UGT2B7 protein, human; D014453:Glucuronosyltransferase