BIOLOGICAL SIGNIFICANCE OF DIVALENT METAL-ION BINDING TO 14-3-3-PROTEINS IN RELATIONSHIP TO NITRATE REDUCTASE INACTIVATION

In this report we address two questions regarding the regulation of ph osphorylated nitrate reductase (pNR; EC 1.6.6.1) by 14-3-3 proteins. T he first concerns the requirement for millimolar concentrations of a d ivalent cation in order to form the inactive pNR:14-3-3 complex at pH 7.5, The second concerns the reduced requirement for divalent cations at pH 6.5, In answering these questions we highlight a possible genera l mechanism involved in the regulation of 14-3-3 binding to target pro teins. We show that divalent cations (e.g. Ca2+, Mg2+ and Mn2+) bind d irectly to 14-3-3s, and as a result cause a conformational change, man ifested as an increase in surface hydrophobicity, A similar change is also obtained by decreasing the pH from pH 7.5 to pH 6.5, in the absen ce of divalent cations, and we propose that protonation of amino acid residues brings about a similar effect to metal ion binding. A possibl e regulatory mechanism, where the 14-3-3 protein has to be ''primed'' prior to binding a target protein, is discussed.