Kinetics of coupled gamma-hydroxybutyrate oxidation and D-glucuronate reduction by an NADP+-dependent oxidoreductase

A highly purified NADP+-dependent oxidoreductase from hamster liver (1) has been found to catalyze a reaction in which the oxidation of gamma-hydroxybutyrate is coupled to the reduction of D-glucuronate. In the coupled reaction, NADP+ is required in catalytic rather than stoichiometric amounts. When the coupled reaction is carried out under limiting conditions for the oxidation of gamma-hydroxybutyrate (i.e. a very low concentration of NADP+ and an inhibitory concentration of NADPH) the rate of gamma-hydroxybutyrate oxidation is determined by the concentration of D-glucuronate and can be stimulated at least 8-fold by D-glucuronate. The kinetics of the oxidation of gamma-hydroxybutyrate have been studied in both the uncoupled and the coupled reaction. In the coupled reaction the reduction of D-glucuronate drives the oxidation of gamma-hydroxybutyrate; the Km for NADP+ is markedly lower than the Km determined in the uncoupled reaction (1.4 X 10(-6) M as compared to 2 X 10(-5) M), and the inhibition by NADPH can be completely overcome. The kinetics of the uncoupled reaction suggest that, unlike many other dehydrogenases, the oxidation of gamma-hydroxybutyrate catalyzed by this enzyme proceeds by a Rapid Equilibrium Random Bi Bi mechanism.