Glutamate Synthase from Escherichia coli

Abstract

Abstract Glutamate synthase catalyzes the TPNH-dependent conversion of α-ketoglutarate and l-glutamine to glutamate (Tempest, D. W., Meers, J. L., and Brown, C. M. (1970) Biochem. J. 117, 405). Levels of glutamate synthase are similar in Escherichia coli grown on 4 mm or 100 mm NH4Cl, but significantly lower levels of enzyme are produced when glutamate replaces NH4Cl as the source of nitrogen. The enzyme was purified to homogeneity from crude extracts of E. coli. Homogeneity was established by cellulose acetate electrophoresis, acrylamide gel electrophoresis, and by sedimentation velocity and sedimentation equilibrium studies. Stability of the purified enzyme is increased in the presence of α-ketoglutarate and 2-mercaptoethanol. The absorption spectrum of the enzyme exhibits maxima at 278, 380, and 440 nm. The purified enzyme contained 7.8 moles of flavin (both FAD and FMN), 38.4 moles of iron, and 30.4 moles of labile sulfide per 800,000 g of protein. Molybdenum was not detected. Enzyme reduced by dithionite was partially (64%) reoxidized by α-ketoglutarate + l-glutamine. The enzyme is highly specific for its substrates. Substrate saturation kinetics is hyperbolic; Km values for TPNH, α-ketoglutarate, and l-glutamine are 7.7, 7.3, and 250 µm, respectively. The pH optimum for catalytic activity is 7.6. Of more than 50 compounds studied only d- and l-aspartate, l-methionine, d-glutamate, and TPN produced 50% inhibition of catalytic activity at concentrations below 10 mm. l-cysteine, l-serine, glycine, l-homoserine, l-glutamate, l-asparagine, l-alanine, and l-histidine also were significant inhibitors. The purified enzyme sediments as a single symmetrical boundary in the analytical ultracentrifuge (s20,w = 20 S). A molecular weight of 800,000 (for v = 0.73) was determined by sedimentation equilibrium and confirmed by gel filtration. On sucrose gradient sedimentation the purified enzyme has a sedimentation coefficient of 20 S but the activity in an ammonium sulfate fraction of crude extract has a sedimentation coefficient of approximately 13 S. The purified protein migrates as a single band during electrophoresis on polyacrylamide gels at pH 7.2 and pH 8.5 and on cellulose acetate at pH 8.4. Enzyme treated with sodium dodecyl sulfate (SDS), urea, or guanidine migrates as two components (mol wt 53,000 and 135,000) on SDS or urea polyacrylamide gel electrophoresis. The enzyme contains 18 half-cystine residues per 200,000 g of protein; one of these is accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the native protein.