Studies on the reaction mechanism of DT diaphorase. Intermediary plateau and trough regions in the initial velocity vs substrate concentration curves

The steady-state kinetics of DT diaphorase [NAD(P)H dehydrogenase (quinone): EC 1.6.99.2]from rat liver has been studied using 2,6-dichloroindophenol as electron acceptor and NADH as electron donor. The υ vs [S]curves revealed intermediary plateau and "trough" regions when NADH and dichloroindophenol were the varying substrates. It has been demonstrated by statistical analysis that equations involving fourth-power terms of the substrate concentrations are able to describe the "trough." These equations have been previously shown in the literature to be capable of describing intermediary plateau regions also. Changes in protein concentration did not affect these curves substantially, whereas changes in pH caused shifts in the positions of their intermediary plateau regions. Increase of the ionic strength by addition of 0.25-0.5 m KCl changed the intermediary plateau region into a "trough" in the υ vs [NADH] plot, whereas the "trough" of the υ vs [dichloroindophenol]plot was transformed into an intermediary plateau. Changes in the temperature of the assay system also led to changes in the kinetic curves. Incubation of the enzyme at 23 °C for 150 min caused disappearance of the intermediary plateau and "trough" regions in the υ vs [NADH]or [dichloroindophenol]plots, respectively. These appeared again after cooling at 0 ° C. The effect of pH, ionic strength, and temperature are discussed within the framework of two main models chosen by statistical analysis: (1) the reaction is catalyzed by a four-site enzyme exhibiting positive and negative cooperativity with respect to the catalytic (kinetic) activity of the sites involved; (2) two enzyme species each possessing two sites are present. These two are either independent or interconnected via a slow isomerization step.