Characterization of glutamate dehydrogenase from the ammonia-oxidizing chemoautotroph Nitrosomonas europaea.

Glutamate dehydrogenase, purified 160-fold by ammonium sulfate precipitation and chromatography on diethylaminoethyl cellulose and Bio-Gel P-300, was characterized as a catalyst of both nicotinamide adenine dinucleotide phosphate reduction and NADPH oxidation at the optimal pH of each reaction (8.7 and 7.7, respectively). In the pH range 7.5 to 8, the rate of enzyme-catalyzed NADPH oxidation is 10 to 15 times greater than the rate of NADPf reduction. The enzyme is specific for NADPH (K,, 4.9 X 10m5 M), a-ketoglutarate (K,, 4.3 mM), NADP+ (K,, 7.9 X 10MG M), and glutamate (K,, 6.7 mM). The K, for NH&l is 16 mM. The oxidation of NADPH was 50% inhibited by 0.5 M NaCl or NH&l, 20 mM D-glUtaUU&, 2 X 10m4 M NADH, 10m3 M NADPf, 4.4 X lOA M NADPH, and 5 pM P-hydroxymercuribenzoate. The reduction of NADP+ was 50% inhibited by 1 m&r NH,Cl, 20 mM D-glutamate or P-alanine, 4 X 10e4 M NADH, and 3 X 10m5 M NADPH. Enzyme activity was not affected by 2 x 10e4 M adenosine or guanosine triphosphate. It appears that in Nitrosomonas the enzyme catalyzes the reaction primarily in the direction of glutamate formation and has a high enough activity to account for all cellular amino nitrogen.