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.
Kremecková
H
H
Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czechoslovakia.
Svrcula
B
B
Mikes
V
V
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