The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense

Abstract

The reactions catalyzed by glutamate synthase from Azospirillum hrasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A . hrasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillurn is specific for the transfer of the 4 s hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent.The data obtained with glutamate synthase from A . hrasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillurn is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Glutamate synthase catalyzes the transfer of the amide group of L-glutamine to the C2 of 2-oxoglutarate, yielding two molecules of glutamate. The reducing equivalents are provided by reduced pyridine nucleotides in microbial glutamate synthases, or by reduced ferredoxin in photosynthetic microorganisms and in the chloroplast enzyme (Vanoni et al., 1991).Glutamate synthase is especially relevant in diazotrophs, since with glutamine synthetase, it forms the major route of ammonia assimilation at low intracellular levels of ammonia. The bacterial enzyme has been purified to homogeneity from a number of enteric bacteria, but only from two diazotrophs, namely Klehsiella aerogenes and Azospirillurn brasilense. The enzyme from A. brasilense shares several properties with that isolated from other bacterial sources (Ratti et al., 1985). It is an iron-sulfur flavoprotein, which contains 1 mol FAD, 1 mol FMN, about 8 atoms non-heme iron and about 8 atoms acidlabile sulfur/l95 kDa protomer. The protomer is formed by two dissimilar subunits of approximately 140 kDa and approximately 55 kDa for the c i and / I subunits, respectively.Bacterial glutamate synthases are specific for NADPH. Lglutamine, and 2-oxoglutarate. At alkaline pH, they can carry

The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-MDa Hexamer by Cryoelectron Microscopy and Its Oligomerization Behavior in Solution

“…In the absence of NaCl, the (␣␤) 6 hexamer prevails, whereas preincubation with NaCl yields the monomeric ␣␤ species. The inclusion of 1 M NaCl in the steady-state kinetic measurements done with the hexameric enzyme form led to a 3-6-fold decrease of the V max value (Table 2) with respect to that measured in the absence of NaCl (15,23,55). The K m values for NADPH and 2-OG increased by 2 orders of magnitude, and that for L-Gln increased 5-10 fold.…”

The mechanism of glutamine-dependent amidotransferases

“…Two sets of figures can be distinguished. For formylglycinamidine synthetase [7] and carbamoyl-P synthetase [39], glutamine is bound first, whereas for glucosamine-6-P synthase [40], asparagine synthetase 5 [41], glutamate synthase [42,43], aminodeoxychorismate synthase [44], and anthranilate synthase [23], the acceptor substrate is bound before glutamine.…”

Kinetic mechanism of ketoreductase activity of prostaglandin F synthase from bovine lung

“…At the same time the turnover number of PGF synthase (4.1 min-' for PGD,) is much less than that of most enzymes, including the previously mentioned flavin-dependent oxidoreductases (e.g. 3,400 min-' for glutamate synthase [6], 90 min-' for dihydropyrimidine dehydrogenase [5]). …”

“…NADH/ NADPH-dependent oxidoreductases obeying the pingpong mechanism usually contain flavin or metal cofactors in their structure. Dihydropyrimidine dehydrogenase from pig liver and glutamate synthase from Azospirillum brasilense, which contain FAD, FMN, and iron-sulfur centers [5,6], and FMN-dependent NADHquinone reductase from E. coli [7] are examples of such enzymes. However, it is remarkable that no such cofactor was reported for PGF synthase.…”

“…Materials [5,6,8,9,12,14,15-'H(N)]PGD, was purchased from DuPont-New England Nuclear. 4-Nitroacetophenone was from Nacalai Tesque, and 4nitrobenzaldehyde and potassium phosphates were from Wako …”