title: Interdomain loops and conformational changes of glutamate synthase as detected by limited proteolysis.
abstract: Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, was subjected to limited proteolysis using trypsin and chymotrypsin, in the absence or presence of its substrates or their analogs. Time-dependent degradation of glutamate synthase alpha and beta subunits, to yield several fragments of different stability, was observed, the alpha subunit being more sensitive than the beta to proteolytic attack. The main sites of proteolytic cleavage were determined by densitometric analysis of the electrophoretic patterns obtained under denaturing conditions and by N-terminal sequencing of the major proteolytic products. These analyses showed that most of the peptide bonds sensitive to the proteases are clustered in two regions of the alpha subunit, outside the proposed substrate and cofactor binding regions of glutamate synthase [Pelanda, R., Vanoni, M. A., Perego, M., Piubelli, L., Galizzi, A., Curti, B. & Zanetti, G. (1993) J. Biol. Chem. 268, 3099-3106]. Therefore, these protease-sensitive sites can be identified as flexible loops, exposed to solvent, connecting adjacent domains of the protein. The presence of the enzyme substrates or their analogs caused significant changes in the proteolytic patterns. NADP+ protected the C-terminal region of glutamate synthase beta subunit from tryptic cleavage, supporting the proposal that it contains the pyridine-nucleotide-binding site. Furthermore, NADP+, and to a lesser extent the glutamine analog L-methionine sulfone, which binds presumably to the N-terminal region of the alpha subunit, altered the sensitivity to proteolysis of the sites of the alpha subunit proposed to be part of links between domains of glutamate synthase. These results show that long-range conformational changes of glutamate synthase occur on binding of its substrates. The study of several NADPH-dependent diaphorase activities of glutamate synthase was also undertaken in order to test if proteolytic fragments of the enzyme retained their ability to transfer electrons from NADPH to synthetic electron acceptors. Although proteolysis yielded partial loss of all enzyme NADPH-dependent reactions, the kinetic analysis showed that the rates of reduction of iodonitrotetrazolium, ferricyanide and dichlorophenolindophenol were at least twofold faster than the rate of the physiological glutamate synthase reaction. These results indicate that enzyme reduction and intramolecular electron transfer are not rate limiting during catalysis of the physiological glutamate synthase reaction.
authors.affiliation: Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.
authors.forename: M A
authors.initials: MA
authors.lastname: Vanoni
authors.forename: A
authors.initials: A
authors.lastname: Mazzoni
authors.forename: P
authors.initials: P
authors.lastname: Fumagalli
authors.forename: A
authors.initials: A
authors.lastname: Negri
authors.forename: G
authors.initials: G
authors.lastname: Zanetti
authors.forename: B
authors.initials: B
authors.lastname: Curti
mesh_terms: D015086:2,6-Dichloroindophenol / Q000378:metabolism; D000595:Amino Acid Sequence; D015806:Azospirillum brasilense / Q000201:enzymology; D002918:Chymotrypsin / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D005970:Glutamate Synthase / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism; D014812:Vitamin K / Q000378:metabolism
chemical_list: D003574:Cytochrome c Group; D010446:Peptide Fragments; D014812:Vitamin K; D009249:NADP; D015086:2,6-Dichloroindophenol; D005970:Glutamate Synthase; D008058:Dihydrolipoamide Dehydrogenase; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin