Regulation of microsomal enzymes by phospholipids. V. Kinetic studies of hepatic uridine diphosphate-glucuronyltransferase.

A bisubstrate kinetic analysis of UDP-glucuronyltransferase (EC 2.4.1.17) has been carried out in forward and reverse directions with p-nitrophenol as aglycone. Reciprocal plots of initial rates of activity indicated that the kinetics followed a sequential mechanism. Product inhibition studies, using UDP and p-nitrophenylglucuronide as inhibitors of the forward reaction, gave a pattern of two competitive and two noncompetitive inhibitions, compatible with a rapid equilibrium, random order kinetic mechanism, or an ordered mechanism of the Theorell-Chance type. Isotope exchange experiments, however, excluded an ordered mechanism. Comparison of the kinetic parameters for the forward and reverse directions showed that the rate at V,,, is 2-fold greater for the reverse than for the forward reaction. At finite substrate concentrations, however, the forward reaction is favored because of the loo-fold higher affinity of the enzyme for p-nitrophenol than for its glucuronide. It was also observed that high concentrations of p-nitrophenol and o-aminophenol have nonspecific activating effects on UDP-glucuronyltransferase. The importance of these findings for the design and interpretation of kinetic experiments is discussed.