Cobalt(III) affinity-labeled aspartokinase. Formation of substrate and inhibitor adducts.
The kinase active site of the aspartokinase-homoserine dehydrogenase enzyme complex of Excherichia coli has been affinity labeled both with substrates aspartate and adenosine triphosphate and feedback inhibitor threonine. Co(III) exchange-inert adducts of aspartokinase and inhibitor or substrates were produced in situ by oxidation of Co(II) with H2O2. Emzyme-Co(III)-adenosine 5'-triphosphate (ATP), enzyme-Co(III)-aspartate, and enzyme-Co(III)-threonine ternary adducts were produced in this manner. The formation of the enzyme-Co(III)-threonine adduct leads us to conclude that threonine inhibits the kinase activity of this enzyme complex by binding in the first coordination sphere of the catalytic metal ion cofactor, a conclusion which is consistent with evidence derived from previous nuclear magnetic resonance data obtained in this laboratory. The quaternary adducts formed by H2O2 oxidation in the presence of aspartokinase, Co(II), ATP, aspartate, and threonine comprised a mixture of both ezyme-Co(III)-ATP-aspartate and enzyme-Co(III)-ATP-threonine adducts. The formation of the quaternary aspartate-containing adduct was unexpected, since the presence of threonine was expected to prevent access of the aspartate to the active site; most significantly however, the the sum of the numbers of aspartate plus threonine molecules incorporated per active site is one. We believe that this shows direct steric overlap between the metal-adjacent binding sites for aspartate and threonine. Aspartate or threonine can not occupy the kinase active site simultaneously; this conclusion is consistent with the direct competitive inhibition of aspartate by threonine observed in steady-state kinetic studies.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D001222:Aspartate Kinase / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism*; D001225:Aspartokinase Homoserine Dehydrogenase / Q000378:metabolism*; D001665:Binding Sites; D001894:Borohydrides / Q000494:pharmacology; D003035:Cobalt / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006861:Hydrogen Peroxide / Q000378:metabolism; D008623:Mercaptoethanol / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D013912:Threonine / Q000494:pharmacology*
D000345:Affinity Labels; D001894:Borohydrides; D009097:Multienzyme Complexes; D013912:Threonine; D001224:Aspartic Acid; D003035:Cobalt; D008623:Mercaptoethanol; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D010770:Phosphotransferases; D001222:Aspartate Kinase; D001225:Aspartokinase Homoserine Dehydrogenase; D004229:Dithiothreitol