Ammonia utilization by a proposed bacterial pathogen in human periodontal disease, Capnocytophaga ochracea.
Capnocytophaga ochracea strain 25 was originally isolated from a patient with severe juvenile periodontitis. An NAD-specific glutamate dehydrogenase (GDH) (EC 1.4.1.2.) was found in cell-free extracts from this organism. The NADH-dependent reductive, or ammonia-assimilating activity (NADH-GDH), of the enzyme was 8-10-fold higher than its NAD-dependent oxidative, or ammonia-releasing activity (NAD-GDH), suggesting that the primary physiological role of the GDH is ammonia-fixing. Capnocytophaga ochracea GDH was purified approximately 39-fold by a rapid, single-step purification procedure using DEAE-cellulose (DE52) ion-exchange column chromatography which gave 90 per cent recovery of total enzyme units. Paper chromatography of an NADH-GDH assay mixture containing the partially purified enzyme showed that glutamate was, indeed, a product of the ammonia-assimilating reaction. The pH optimum for the NAD-GDH reaction was 9.0; that for the NADH-GDH reaction was 7.5. Although a number of mono- and divalent cations were tested, none had a large effect on either NAD-GDH or NADH-GDH activity. The NAD-GDH reaction showed a hyperbolic kinetic response to glutamate and NAD and the Km values for glutamate and NAD were 2.44 and 0.083 mM respectively. The kinetic response of the NADH-GDH reaction to NADH, alpha-ketoglutarate and ammonium chloride also obeyed Michaelis-Menten kinetics and their respective Km values were 0.069, 1.44 and 3.33 mM. Of a number of biologically-active compounds tested for their ability to modulate GDH activity, only ADP and NAD exerted much effect. The NADH-GDH activity showed a negative hyperbolic kinetic response to both ADP and NAD and Dixon plot-analysis of the NAD and ADP saturation data gave Ki values for ADP and NAD of 4.0 and 0.46 mM respectively. Both NAD and ADP appeared to exert their negative effects on NADH-GDH activity by completely inhibiting the binding of the reduced coenzyme, NADH, to the enzyme.
D000641:Ammonia / Q000378:metabolism*; D001424:Bacterial Infections; D002206:Capnocytophaga / Q000378:metabolism*; D041964:Cytophagaceae / Q000378:metabolism*; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD; D010518:Periodontitis / Q000209:etiology; D013379:Substrate Specificity
D009243:NAD; D000641:Ammonia; D005969:Glutamate Dehydrogenase