Studies on the DT-diaphorase-catalysed reaction employing quinones as substrates: evidence for a covalent modification of DT-diaphorase by tetrachloro-p-benzoquinone.
In this study, the kinetic parameters, V(max) and K(m), 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 V(max)/K(m) 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-benzoquinone 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.
Osman
Ahmed M
AM
Institute for Inland Water Management and Waste-water Treatment, P.O. Box 17, 8200 AA Lelystad, The Netherlands. a.osman@riza.rws.minvenw.nl
Boeren
Sjef
S
D000818:Animals; D016227:Benzoquinones / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D002384:Catalysis / Q000187:drug effects; D002417:Cattle; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D016227:Benzoquinones; D012710:Serum Albumin, Bovine; D016660:NAD(P)H Dehydrogenase (Quinone)