Author(s): Uchaipichat V, Galetin A, Houston JB, Mackenzie PI, Williams JA,
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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.
This article was published in Mol Pharmacol
and referenced in Pharmaceutica Analytica Acta