Cobalt(III) affinity-labeled aspartokinase. Formation of substrate and inhibitor adducts

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Abstract
The kinase active site of the aspartokinase[EC 2.7.2.4]-homoserine dehydrogenase [EC 1.1.1.3] enzyme complex of Escherichia coli was affinity labeled with substrates aspartate and ATP 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. Enzyme-Co(III)-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 indicates that threonine inhibits the kinase activity of this enzyme complex by binding in the 1st coordination sphere of the catalytic metal ion cofactor, which is consistent with previous NMR data. The quaternary adducts formed by H2O2 oxidation in the presence of aspartokinase, Co(II), ATP, aspartate and threonine comprised a mixture of enzyme-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 should prevent access of the aspartate to the active site; but the sum of the numbers of aspartate plus threonine molecules incorporated per active site is one. Thus, a direct steric overlap between the metal-adjacent binding sites for aspartate and threonine occurs. 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.

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