A note on the inhibition of DT-diaphorase by dicoumarol

Abstract

The participation of DT-diaphorase or NAD(P)H:(quinone acceptor) oxidoreductase (E.C.1.6.99.2) in metabolism or in events leading to toxicity is often implied on the basis of the inhibitory effects of dicoumarol. DT-diaphorase functions via a ping pong bi-bi kinetic mechanism involving oxidized and reduced flavin forms of the free enzyme. Dicoumarol, a potent (K 1 = 10 nM) inhibitor, binds to the oxidized form of the enzyme, competitively versus reduced pyridine nucleotide. Inhibition is effectively complete at 1 μM dicoumarol in typical studies using DCPIP, one of the best known substrate for the enzyme, as electron acceptor. The antitumor quinone Diaziquone (AZQ) is a poor substrate for DT-diaphorase relative to DCPIP, but effective inhibition of its reduction requires ten-fold higher concentrations of dicoumarol than for inhibition of DCPIP reduction under otherwise similar conditions. The variable inhibition of DT-diaphorase by dicoumarol dependent on the efficiency of the electron acceptor can be explained on the basis of the complete rate equation describing its ping pong type kinetic mechanism. Thus, the concentration of dicoumarol used to inhibit DT-diaphorase must be chosen carefully and consideration should be given to the efficiency of the electron acceptor. The absence of an inhibitory effect using low doses of dicoumarol cannot rule out a reaction mediated by DT-diaphorase. Although higher doses of dicoumarol may be required to inhibit DT-diaphorase mediated metabolism of less efficient electron acceptors, the use of such doses in cells may also effect biochemical processes other than DT-diaphorase and should be approached with caution.