Studies on the DT-diaphorase-catalysed reaction employing quinones as substrates: evidence for a covalent modification of DT-diaphorase by tetrachloro-p-benzoquinone
Osman, A.M. ; Boeren, J.A. - \ 2004
Chemico-Biological Interactions 147 (2004)1. - ISSN 0009-2797 - p. 99 - 108.
one-electron - rat-liver - acceptor oxidoreductase - mechanism - reductase - nitrobenzimidazoles - cytotoxicity - conversion - protein - nad(p)h
In this study, the kinetic parameters, Vmax and Km, of rat liver DT-diaphorase were determined for a series of p-benzoquinones, with methyl, methoxy, cyano, hydroxy and halo substituents. The results show that there is no correlation between the experimentally determined rates of p-benzoquinone reduction by DT-diaphorase and the calculated chemical reactivity of the examined substrates as expressed by the energy of the lowest unoccupied molecular orbital, E(LUMO). However, a reasonable correlation was found between the natural logarithm of Vmax/Km and the partition coefficient of the p-benzoquinones (r=0.81). Furthermore, tetrachloro-p-benzoquinone, one of the tested quinones is shown to be an inhibitor of rat DT-diaphorase. The presence of bovine serum albumin (BSA) in the incubation mixture protects DT-diaphorase against the inactivation by tetrachloro-p-benzoquinone, probably by interacting with the quinone. Maldi-Tof analysis of the incubation mixture of the purified DT-diaphorase and tetrachloro-p-benquinone showed that every subunit of the enzyme shifted about +414 amu, whereas the dimer shifted about +849 amu relative to control values. This indicates a covalent modification of the rat liver DT-diaphorase by tetrachloro-p-benzoquinone.