Figure 3.

GTP-dependence of urease apoprotein activation for the UreD-UreF-UreG-apourease complex. (A) The time dependence of urease apoprotein activation using UreD-UreF-UreG-apourease (0.2 μM) was studied at 37°C in the presence (closed symbols) or absence (open symbols) of 200 μM Mg₂GTP in 100 mM Hepes buffer (pH 8.3) containing 150 mM NaCl, 100 μM NiCl₂, and 100 mM (squares) or 100 μM (circles) bicarbonate. (B) The nucleotide specificity and concentration dependence was examined by monitoring the extent of activation after 1.5 h in buffer containing 100 μM NiCl₂, 100 μM NaHCO₃, and the indicated concentrations of Mg₂GTP (■), Mg₂ATP (▴), Mg₂GDP (●), and Mg₂GMP-PNP (▾). (C) The effect of using a P-loop defective UreG variant (T21A UreG) was assessed. The urease apoproteins in UreD-UreF-UreG-apourease complexes containing the variant protein (0.17 μM; ■) or native UreG (0.2 μM; ●) were activated for 1.5 h in the presence of 100 μM NiCl₂, 100 μM NaHCO₃, and GTP at the concentrations indicated. In all cases, urease activities were measured as previously described. All results are representative of three separate protein preparations.