Glutamate Synthase from Escherichia coli

作者：Richard E. Miller, Earl R. Stadtman · 发表于：Journal of Biological Chemistry · 年份：1972 · DOI：10.1016/s0021-9258(19)44642-5 · 被引用次数：269 · 研究领域：Enzyme Structure and Function、Amino Acid Enzymes and Metabolism、Metabolomics and Mass Spectrometry Studies

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-cys...