Purification and some properties of glutamate dehydrogenase and glutamine synthetase from Paracoccus denitrificans.
The purification and some properties of NADP-dependent glutamate dehydrogenase (GDH) and glutamine synthetase (GS) from the facultatively anaerobic Gram-negative bacterium Paracoccus denitrificans were investigated. The enzymes were purified to homogeneity using a procedure which involved affinity chromatography on Blue Sepharose CL-6B as the major purification step. The recoveries in the purification of GDH and GS were 28% and 64%, respectively. The specific activity of purified GDH was 183 nkat (mg protein)-1 (deaminating reaction). GDH was composed of subunits of molecular mass 47 kDa and the native enzyme was either a tetramer or hexamer. The apparent Km values for L-glutamate, NADP, 2-oxoglutarate, NADPH and ammonia were 1.5 mM, 5.9 microM, 0.47 microM, 12.5 microM and 14 mM, respectively. The specific activity of purified GS was 1125 nkat (mg protein)-1 (transferase reaction). The molecular mass of native GS was 570 kDa; it was composed of 12 subunits of molecular mass 50.1 kDa. The apparent Km values for L-glutamine and hydroxylamine in the transferase reaction were 2.1 and 2.4 mM, respectively; those of ammonia, L-glutamate and ATP in the biosynthetic reaction were 0.03, 1 and 0.17 mM, respectively. After the adenylylation of GS, the Km for L-glutamine and L-glutamate increased and reached the values of 8.0 and 27 mM, respectively. The effects of the changes in GS activity on the ammonia metabolism of Paracoccus denitrificans are discussed.
Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czechoslovakia.
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Kremecková
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Svrcula
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Mikes
D004591:Electrophoresis, Polyacrylamide Gel; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D010231:Paracoccus denitrificans / Q000201:enzymology*
D005969:Glutamate Dehydrogenase; D005974:Glutamate-Ammonia Ligase