Activation and coupling of the glutaminase and synthase reaction of glutamate synthase is mediated by E1013 of the ferredoxin-dependent enzyme, belonging to loop 4 of the synthase domain.
Crystal structures of glutamate synthase suggested that a conserved glutamyl residue of the synthase domain (E1013 of Synechocystis sp. PCC 6803 ferredoxin-dependent glutamate synthase, FdGltS) may play a key role in activating glutamine binding and hydrolysis and ammonia transfer to the synthase site in this amidotransferase, in response to the ligation and redox state of the synthase site. The E1013D, N, and A, variants of FdGltS were overproduced in Escherichia coli cells, purified, and characterized. The amino acyl substitutions had no effect on the reactivity of the synthase site nor on the interaction with ferredoxin. On the contrary, a dramatic decrease of activity was observed with the D (approximately 100-fold), N and A (approximately 10,000-fold) variants, mainly due to an effect on the maximum velocity of the reaction. The E1013D variant showed coupling between glutamine hydrolysis at the glutaminase site and 2-oxoglutarate-dependent L-glutamate synthesis at the synthase site, but a sigmoid dependence of initial velocity on L-glutamine concentration. The E1013N variant exhibited hyperbolic kinetics, but the velocity of glutamine hydrolysis was twice that of glutamate synthesis from 2-oxoglutarate at the synthase site. These results are consistent with the proposed role of E1013 in signaling the presence of 2-oxoglutarate (and reducing equivalents) at the synthase site to the glutaminase site in order to activate it and to promote ammonia transfer to the synthase site through the ammonia tunnel. The sigmoid dependence of the initial velocity of the glutamate synthase reaction of the E1013D mutant on glutamine concentration provides evidence for a participation of glutamine in the activation of glutamate synthase during the catalytic cycle.
Dossena
Laura
L
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.
Curti
Bruno
B
Vanoni
Maria A
MA
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000641:Ammonia / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D046939:Synechocystis / Q000201:enzymology
D018698:Glutamic Acid; D000641:Ammonia; D000594:Amino Acid Oxidoreductases; C042233:glutamate synthase (ferredoxin)