title: Nuclear-magnetic-relaxation studies of the interaction of inhibitor with the threonine-sensitive aspartokinase of Escherichia coli.
abstract: The nature of the feedback inhibition of the bifunctional enzyme, aspartokinase I-homoserine dehydrogenase I of Escherichia coli was studied using 13C nuclear magnetic resonance (NMR). Since aspartokinase is activated by Mn(II), the interaction of the inhibitor L-threonine (specifically enriched to 90% 13C in the carboxyl carbon) with the metal-enzyme complex was studied. Spin-lattice (T1) and spin-spin (T2) relaxation times were determined by the partially relaxed Fourier transform method and line-width measurements respectively at 20 MHz. The pronounced broadening of the DL-threonine carboxyl peak in the presence of the Mn(II)-enzyme complex indicates that an L-threonine binding site is close to the metal binding site of the kinase active site. The non-identity of (T1)*M and (T2)*M indicates that conditions of fast exchange prevail. The (T1)*M/(T2)*M ratio was used to estimate a correlation time of 2.0 ns for the dipolar interaction at 25 degrees C. An estimate for the distance between Mn(II) and the threonine carboxyl carbon of 4.4 A (0.44 nm) was obtained. This 13C NMR study has thus located one of the two classes of threonine regulatory sites which exist per subunit; the threonine site identified here is at the aspartokinase active site, adjacent to the catalytic metal site.
mesh_terms: D001222:Aspartate Kinase / Q000037:antagonists & inhibitors*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005246:Feedback; D005583:Fourier Analysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000494:pharmacology; D008433:Mathematics; D010770:Phosphotransferases / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature; D013912:Threonine / Q000494:pharmacology*
chemical_list: D013912:Threonine; D008345:Manganese; D010770:Phosphotransferases; D001222:Aspartate Kinase