BOVINE INOSITOL MONOPHOSPHATASE - LIGAND-BINDING TO PYRENE-MALEIMIDE-LABELED ENZYME

Inositol monophosphatase can be modified at two sites by pyrene maleim ide. These sites have been identified as Cys141 and Cys218. Stoichiome tric addition of pyrene maleimide allows the sole modification of Cys2 18. The fluorescence of the pyrene moiety on the modified protein can be excited directly or by resonance energy transfer. The fluorescence properties of the pyrene group on Cys218 allows the interaction of lig ands with the enzyme to be monitored. This feature has allowed dissoci ation constants for various metal ions to be determined and allowed th e formation of various enzyme/ligand complexes to be observed. These s tudies have demonstrated that Mg2+ is required to support P-i binding and that Li+ interacts with a post-catalytic complex which is only for med in the forward reaction.