STABLE MONOMERIC FORM OF AN ORIGINALLY DIMERIC SERINE PROTEINASE-INHIBITOR, ECOTIN, WAS CONSTRUCTED VIA SITE-DIRECTED MUTAGENESIS

Ecotin, a homodimer protein of E. coli, is a unique member of canonica l serine proteinase inhibitors, since it is a potent agent against a v ariety of serine proteinases having different substrate specificity, M onomers of ecotin are held together mostly by their long C-terminal st rands that are arranged as a two-stranded antiparallel beta-sheet in t he functional dimer, One ecotin dimer can chelate two proteinase molec ules, each of them bound to both subunits of ecotin at two different s ites, namely the specific primary and the non-specific secondary bindi ng sites, In this study the genes of wild type ecotin and its Met(84)A rg P1 site mutant were truncated resulting in new forms of ecotin that lack 10 amino acid residues at their C-terminus, These mutants do not dimerize spontaneously, though in combination with trypsin they assem ble into the familiar heterotetramer, Our data suggest that this heter otetramer exists even in extremely diluted solutions, and the interact ion, which is responsible for the dimerization of ecotin, contributes to the stability of the heterotetrameric complex.