Magnetic resonance studies of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium

The binding of Mn 2+ to the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium was examined by electron paramagnetic resonance studies. Two types of binding sites were observed: one to two tight sites with a dissociation constant of 3–5 μ m and five to six weaker sites with a dissociation constant of 40–70 μ m. The activator constant for Mn 2+ was found to be 9 μ m for the glutamine-linked anthranilate synthetase activity and 4 μm for the phosphoribosyltransferase activity. These values are both in the range of the dissociation constant for the tight sites. Water proton relaxation rate measurements showed that the binary enhancement values for both classes of sites were equivalent, ϵ b = 10.7 ± 2.0. The addition of chorismate to the Mn 2+-enzyme complexes when predominantly the tight Mn 2+ sites were occupied resulted in a large decrease in the observed enhancement ( ϵ T = 2.0). Addition of 5-phosphoribosyl-1-pyrophosphate to the enzyme-Mn 2+ complexes caused large decreases in the water proton relaxation rate ( ϵ T = 1.5) when tight or tight plus weaker Mn 2+ sites were occupied. No changes in the water proton relaxation rate were observed when glutamine, pyruvate, or anthranilate were added; a small decrease was observed when enzyme-Mn 2+ was titrated with tryptophan. Tryptophan significantly altered the effect of the binding of chorismate but not of 5-phosphoribosyl-1-pyrophosphate. The effect of tryptophan on the water proton relaxation rate of a Mn 2+-enzyme-chorismate complex using a variant enzyme complex which is tryptophan hypersensitive ( P. D. Robison, and H. R. Levy, 1976, Biochim. Biophys. Acta. 445, 475–485) occurred at lower concentrations than for the normal enzyme complex. The uncomplexed anthranilate synthetase subunit was titrated with Mn 2+ and found to have one to two binding sites with a dissociation constant of 300 ± 100 μ m. This dissociation constant is much larger than the activator constant for Mn 2+ for uncomplexed anthranilate synthetase which was determined to be 4 μ m. These results indicate that the Mn 2+-binding sites on anthranilate synthetase are altered when the enzyme complex is formed and that both chorismate and 5-phosphoribosyl-1-pyrophosphate interact closely with enzyme-bound Mn 2+ or cause a large effect upon its environment.