Modulation of the number of ligand binding sites of UDP-glucuronyltransferase by the gel to liquid-crystal phase transition of phosphatidylcholines.
The kinetics of a pure, delipidated form of microsomal UDP-glucuronyltransferase is non-Michaelis-Menten when the enzyme is reconstituted into unilamellar vesicles of phosphatidylcholine that are in a gel phase. Double reciprocal plots of velocity as a function of the concentration of UDP-glucuronic acid show a downward curvature under these conditions. Binding studies indicate that the basis for the kinetic pattern is the presence of one high affinity and one low affinity binding site for UDP-glucuronic acid. The two classes of binding sites seem to be generated by the presence of two subunits that bind UDP-glucuronic acid within a single molecule of UDP-glucuronyltransferase. Melting the phospholipids from the gel phase to the liquid-crystal phase is associated with a switch from non-Michaelis-Menten to Michaelis-Menten kinetics for UDP-glucuronyltransferase. Binding studies for interaction of UDP-glucuronic acid with enzyme present in a liquid-crystal lipid phase indicate that the two binding sites for UDP-glucuronic acid do not become identical in this setting. Instead, one of the sites becomes nonfunctional. Binding studies carried out with UDP as ligand lead to similar results. There is a high affinity and a low affinity site for UDP when enzyme is reconstituted into a phospholipid bilayer in a gel phase. There is only one UDP binding site per holoenzyme when enzyme is reconstituted into a phospholipid bilayer in a liquid-crystal phase. Delipidated enzyme or enzyme reconstituted with lysophosphatidylcholine displays Michaelis-Menten kinetics. Binding studies show that these forms have only one binding site for UDP-glucuronic acid per holoenzyme. However, they have two nonidentical binding sites for binding of UDP. Thus, the physical properties of its phospholipid milieu influence the number of functional binding sites of UDP-glucuronyltransferase.
D000818:Animals; D001665:Binding Sites; D005782:Gels; D014453:Glucuronosyltransferase / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D010713:Phosphatidylcholines; D013552:Swine; D013696:Temperature; D014530:Uridine Diphosphate / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism
D005782:Gels; D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D014535:Uridine Diphosphate Glucuronic Acid; D014530:Uridine Diphosphate; D014453:Glucuronosyltransferase