Figure 3.

The AbNadE2 activity is down-regulated by NAD⁺ and GlnZ relives AbNadE2 from NAD⁺ inhibition. NadE2 discontinuous assays were performed to measure the formation of PP_i in the presence of 4 mm l-glutamine, 4 mm ATP, and 2 mm NaAD. Data were plotted as relative percent activity considering the reaction without NAD⁺ as 100% activity. A, effect of redox metabolites, when indicated 2.5 mm NAD⁺ or 1 mm of NADH, NADP⁺ or NADPH were added. An asterisk indicates statistically significant difference (t test, p < 0.01), n = 3. B, reactions were carried out in the presence of 100 nm NadE2Ab (monomer), GlnZ was added at 2 μm (trimer) when indicated. Reactions without GlnZ were contained 2 μm BSA. The calculated AbNadE2 NAD⁺ K_i was 1 mm and 2.5 mm in the absence and presence of GlnZ, respectively. Data were analyzed and the IC₅₀ were calculated by nonlinear regression in GraphPad Prism 7. S. D. from triplicate experiments are indicated by error bars.