MOLECULAR AND KINETIC CHARACTERIZATION OF GLUTAMATE SYNTHASE FROM THEPHOTOTROPHIC BACTERIUM RHODOBACTER-CAPSULATUS E1F1

Glutamate synthase (GOGAT) from the phototrophic non-sulphur purple ba cterium Rhodobacter capsulatus E1F1 has been purified to electrophoret ic homogeneity by affinity chromatography. The native protein consiste d of two different subunits of 175 and 53 kDa and contained 4 mol FAD, 4 mol iron and 4 mol labile sulphide per mol of dimer enzyme. The enz yme used NADPH as the electron donor and was inhibited by iron-chelati ng and thiol group reagents. GOGAT exhibited NAD(P)H-diaphorase activi ty which used sodium ferricyanide, cytochrome c and dichlorophenol ind ophenol as alternative electron accepters. By contrast, glutaminase ac tivity was not detected in purified GOGAT. The amino acid composition was quite different from that of other bacterial GOGATs, and the prote in presented different aggregation states depending on the ionic stren gth. Two major multimeric active species with Stokes' radii of 6.18 an d 7.32 nm could be separated by gel-filtration of protein solutions ma de in 0.5 M-KCl, whereas in the absence of salt, the maximal GOGAT act ivity corresponded to an oligomer with Stokes' radius of 6.80 nm. The enzyme exhibited apparent negative cooperativity for glutamine, and wa s competitively inhibited by L-glutamate and NADP(+).