Studies by affinity chromatography on the NAD(P)H and FAD sites of nitrate reductase from ankistrodesmusbraunii

Abstract

Both native NAD(P)H-nitrate reductase (E.C. 1.6.6.2.) from Ankistrodesmus braunii and the FAD-depleted enzyme are adsobed on blue dextran—Sepharose at two different sites. The binding of the native enzyme involves the NAD(P)H active site, while that of the deflavoenzyme uses the FAD site. The holoenzyme can be specifically eluted by 0.5 m M NAD(P)H, but the elution of the deflavoenzyme was achieved only by the simultaneous addition of 1 m M FAD and 0.5 M KCl to the washing buffer. Incubation of native or flavin-free nitrate reductases with p-hydroxymercuribenzoate prevents adsorption of both types of enzyme on blue dextran—Sepharose, which indicates the presence of sulphydryl groups in the sites for NAD(P)H and FAD. Binding studies of the holoenzyme on three different kinds of NAD-Agarose indicate that the NAD(P)H-domain is acting as a crevice in which the nicotinamide ring of the nucleotide should be placed at the bottom. In addition, the structure of the binding site for FAD seems to be similar to that for NAD(P)H.