Properties and Reaction Mechanism of DT Diaphorase from Rat Liver

Abstract

Abstract DT diaphorase was purified to homogeneity from rat liver and characterized. The molecular weight of the enzyme was calculated to be 5.0 ± 0.06 x 104 from sedimentation equilibrium experiments and to be 4.8 x 104 by thin layer gel filtration method using Sephadex G-200. The identity of FAD as a prosthetic group was confirmed by d-amino acid oxidase test. It was found that 1 mole of FAD was present per mole of enzyme. The interaction of the enzyme with NADPH and K3 in the presence and absence of bovine serum albumin and dicumarol were also studied by steady state and stopped flow kinetic methods, together with binding experiments using [14C]K3 and [14C]dicumarol. The probable reaction sequences of the enzyme, including the pyridine nucleotide-dependent reduction of K3 mainly by a ping-pong type mechanism with a rate-limiting step at the point of dissociation of the enzymereduced K3 complex, are proposed. There is evidence that the native form of enzyme has two independent binding sites for K3 and dicumarol in its active center, forming the partially active enzyme-K3 complex reducible by NADPH but not oxidizable by K3 and the fully inactive enzyme-dicumarol complex not reducible by NADPH. The reduced species is capable of binding only K3. It was also demonstrated that bovine serum albumin competes with the native enzyme for K3 and dicumarol, probably through the ability of this simple protein to associate with these substances, preventing the formation of enzyme-K3 and enzyme-dicumarol complexes, and leading to the apparent increase in enzyme activity.