1887

Abstract

Glutamate synthase (GOGAT) from the phototrophic non-sulphur purple bacterium ElFl has been purified to electrophoretic homogeneity by affinity chromatography. The native protein consisted 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 enzyme used NADPH as the electron donor and was inhibited by iron-chelating and thiol group reagents. GOGAT exhibited NAD(P)H-diaphorase activity which used sodium ferricyanide, cytochrome and dichlorophenol indophenol as alternative electron acceptors. By contrast, glutaminase activity was not detected in purified GOGAT. The amino acid composition was quite different from that of other bacterial GOGATs, and the protein presented different aggregation states depending on the ionic strength. Two major multimeric active species with Stokes’ radii of 6·18 and 7·32 nm could be separated by gel-filtration of protein solutions made in 0·5 -KCl, whereas in the absence of salt, the maximal GOGAT activity corresponded to an oligomer with Stokes radius of 6·80 nm. The enzyme exhibited apparent negative cooperativity for glutamine, and was competitively inhibited by -glutamate and NADP.

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1993-12-01
2022-01-23
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