Magnetic resonance and kinetic studies of the partial complex and Component I subunit forms of Salmonella typhimurium anthranilate synthase.
Metal ion interactions of the monofunctional partial complex of Salmonella typhimurium anthranilate synthase were investigated using kinetic, NMR, and EPR methods. Mn2+ activates AS-partial complex in place of Mg2+, with a Km of 0.08 microM for Mn2+ and of 3.5 microM for Mg2+ in glutamine-dependent anthranilate synthase activity. The kinetics indicated that the metal interacts at the active site with chorismate, not glutamine. EPR and NMR water proton relaxation rate (PRR) studies supported this conclusion. EPR binding analysis showed that chorismate dramatically tightens Mn2+ binding by the partial complex. PRR experiments indicated that stoichiometric amounts of chorismate cause a substantial decrease in the enhancement of water relaxation by Mn2+, while millimolar amounts of glutamine have no effect. Analysis of the frequency dependence of water proton relaxation rates yielded dipolar correlation times of 2.5 x 10(-9) s and 4.1 x 10(-9) s for the Mn2+-partial complex and Mn2+-partial complex-chorismate complexes, respectively. These studies also indicated that chorismate binding reduces the number of fast-exchanging water molecules on enzyme-bound Mn2+ from 1 to 0.25. PRR experiments with the native bifunctional anthranilate synthase-phosphoribosyltransferase enzyme indicated the existence of additional Mn2+-binding sites which presumably function to activate the phosphoribosyltransferase activity of the Component II subunit.
Summerfield
A E
AE
Department of Chemistry, University of Virginia, Charlottesville 22901.
Bauerle
R
R
Grisham
C M
CM
D000878:Anthranilate Synthase / Q000378:metabolism*; D001665:Binding Sites; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000494:pharmacology; D008433:Mathematics; D012486:Salmonella typhimurium / Q000201:enzymology*
D008345:Manganese; D000878:Anthranilate Synthase; D008274:Magnesium