Properties and reaction mechanism of DT diaphorase from rat liver.

DT diaphorase was purified to homogeneity from rat liver and characterized. The molecular weight of the enzyme was calculated to be 5.0 h 0.06 x lo4 from sedimentation equilibrium experiments and to be 4.8 x IO4 by thin layer gel filtration method using Sephadex G-200. The identity of FAD as a prosthetic group was confirmed by o-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 f14C]Ks 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 enzymethe yellow color in oxidized Aavoprotein after adding excess NAD(P)H, a marked increase in enzyme activity produced by bovine serum albumin, and a powerful specific inhibition by dicumarol (2, 4, 6). Further clarification of these aspects, however, has not been accomplished, since no precise react’ion mechanism of this enzyme had been established. In this paper we report some physicochemical properties of DT diaphorase purified from rat liver, together with steady state and stopped flow kinetic studies of the interaction of the enzyme with NADPH and Ka in the presence and absence of serum albumin as well as of dicumarol. It is proposed that DT diaphorase catalyzes the pyridine nucleotide-dependent reduction of Ka mainly by a ping-pong type mechanism in which the oxidized enzyme is reduced by NADPH to produce the free reduced species. This type of mechanism is indicated schematically in Equations 1, 2, 3, and 4.