| Abstract: | Reduced pyridine nucleotide dependent glutamate synthase [L-glutamate: NADP+ oxidoreductase (transaminating); EC 1.4.1.13] was purified to homogeneity from Bacillus subtilis PCI 219. The molecular weight of the enzyme was 210,000, and the enzyme was composed of two nonidentical subunits with molecular weights of 160,000 and 56,000. The absorption and CD spectra of the enzyme indicated that the enzyme is an iron-sulfur flavoprotein. The enzyme was found to contain 1:1:7.4:8.7 mol of FMN, FAD, iron atoms, and acid-labile sulfur atoms per mol (MW 210,000). EPR measurements of the NADPH-reduced enzyme at 77K revealed the formation of a stable flavin semiquinone intermediate; however, none of the signals originating from the iron-sulfur cluster was observed. Still at 4.2K the EPR signals in the region of g = 2, which may originate from the paramagnetic iron-sulfur cluster, were clearly observed for both the isolated and dithionite-reduced states of the enzyme. The enzyme exhibited a wide coenzyme specificity, and either NADPH or NADH could be used as electron donor, although the latter was less effective. The enzyme activity was also expressed when ammonium chloride was substituted for L-glutamine. The optimum pHs for NADPH-Gln-, NADH-Gln-, and NADPH-NH3-dependent reactions were 7.8, 6.9, and 9.4, respectively. The apoenzyme exhibited substantial inactivation of the Gln-dependent activities but still retained the NH3-dependent activities. Enzyme reduction-oxidation experiments, initial velocity experiments, and product inhibition patterns revealed that both the NADPH-Gln- and NADH-Gln-dependent reactions coincided with the two-site ping-pong uni-uni bi-bi kinetic mechanism, while the NADPH-NH3-dependent reaction deviated from Michaelis-Menten kinetics. The Gln-dependent activities were inhibited by several TCA cycle members, especially L-malate and fumarate, as well as L-methionine-SR-sulfoximine, pyridoxal-5'-phosphate, and pCMB. The regulation of the glutamate synthase, glutamine synthetase [EC 6.3.1.2], and glutamate dehydrogenase [EC 1.4.1.3] activities was examined with cultures of cells grown with various nitrogen and carbon sources. |
