THIOL ACTIVATION OF ENDOPEPTIDASE EC-3.4.24.15 - A NOVEL MECHANISM FOR THE REGULATION OF CATALYTIC ACTIVITY

Endopeptidase EC 3.4.24.15 (EP24,15) is a thermolysin-like metalloendo peptidase involved in the regulated metabolism of a number of neuropep tides. Unlike other thermolysin-like peptidases EP24.15 displays a uni que thiol activation, a mechanism that is not clearly understood, In t his study we show that both recombinant and tissue-derived EP24,15 are activated up to 8-fold by low concentrations (0.1 mM) of dithiothreit ol, Additionally, under non-reducing conditions, recombinant and nativ e EP24,15 forms multimers that can be returned to the monomeric form b y reduction, We have also shown that competitive inhibitor binding occ urs only to the monomeric form, which indicates that catalytic site ac cess is restricted in the multimeric forms, Through systematic site-di rected mutagenesis we have identified that cysteine residues 246, 253, and possibly 248 are involved in the formation of these multimers, Fu rthermore, both a double mutant (C246S/C253S) and a triple mutant (C24 6S/C248S/C253S) are fully active in the absence of reducing agents, as measured by both inhibitor binding and hydrolysis, The formation and disruption of disulfide bonds involving these cysteine residues may be a mechanism by which EP24,15 activity is regulated through changes in intra- and extracellular redox potential.