Interaction of uridine 5′-diphosphoglucuronic acid with microsomal UDP-glucuronosyltransferase in primate liver: the facilitating role of uridine 5′-diphospho- N-acetylglucosamine

Abstract

Cytosolic uridine 5′-diphosphoglucuronic acid is the essential cosubstrate for all hepatic microsomal UDP-glucuronosyltransferase-mediated reactions. Uridine 5′-diphospho- N-acetylglucosamine (UDP-GlcNAc) has been implicated as an activator of UDP-glucuronosyltransferases in vivo, acting either as an allosteric effector or by enhancing access of uridine 5′-diphosphoglucuronic acid to the enzyme. To delineate the interaction of uridine 5′-diphosphoglucuronic acid with microsomal UDP-glucuronosyltransferase and the facilitating role of UDP-GlcNAc, we analyzed bilirubin UDP-glucuronosyltransferase kinetics in microsomes prepared from monkey liver ( Macaca fascicularis). Initial rates of bilirubin glucuronide formation were determined by radiochemical assay over a range of uridine 5′-diphosphoglucuronic acid concentrations (0–60 mM), in native microsomes with or without UDP-GlcNAc, or in detergent (digitonin)-pretreated membranes with UDP-GlcNAc. For native microsomes in the absence of UDP-GlcNAc, fitting the data to each of two mathematical models yielded behavior consistent with a single-site model ( K m 2.8 mM). In contrast, in the presence of a physiologic concentration (1 mM) of UDP-GlcNAc, analysis of the data excluded the single-site model and was indicative of a non-interactive, two-site (or process) model, characterized by a high-affinity site ( K m 0.14 mM) in addition to the low-affinity site. Following detergent-treatment of microsomal membranes, the data were again most consistent with a single low-affinity site. These findings demonstrate that UDP-GlcNAc enhances glucuronidation in primate liver, by altering the kinetic interaction of uridine 5′-diphosphoglucuronic acid and UDP-glucuronosyltransferase (i.e., emergence of a second, high-affinity site), and are compatible with the hypothesis that UDP-GlcNAc facilitates the transport of uridine 5′-diphosphoglucuronic acid from the cytosol to the enzyme via a microsomal membrane permease or transporter.