Catalytic properties of Ankistrodesmus braunii nitrate reductase

Abstract

Woolf plots of the initial reaction velocities at varying concentrations of substrates have been performed to determine the substrates affinity for the purified A. branuii NAD(P)H-nitrate reductase complex. p-Hydroxymercuribenzoate, cyanide or azide inhibits the reduction of nitrate by NAD(P)H catalysed by this enzyme. The inhibition of p-hydroxymercuribenzoate is localised in the first activity of the complex, i.e. NAD(P)H-cytochrome c reductase, and this inhibition is specifically protected by NADH, but not by NADPH. Cyanide or azide inhibits the terminal activity of the complex, i.e. reduced methyl viologen-nitrate reductase. Lineweaver-Burk plots of the initial velocities, at different concentrations of NADH and nitrate, and product inhibition patterns show an iso ping pong bi bi steady state kinetic mechanism, with isomeration of the enzymatic form which binds NADH, for the NADH-nitrate reductase activity.