CHANGING THE INHIBITORY SPECIFICITY AND FUNCTION OF CUCURBITA-MAXIMA TRYPSIN INHIBITOR-V BY SITE-DIRECTED MUTAGENESIS

Cucurbita maxima trypsin inhibitor-V (CMTI-V) is also a specific inhib itor of human blood coagulation factor beta-factor XII(a). A recombina nt version of CMTI-V has allowed probing of roles of individual amino acid residues including the reactive site residue, lysine (P1), by sit e-directed mutagenesis. The K44R showed at least a 5-fold increase in inhibitory activity toward human beta-factor XII(a), while there was n o change toward bovine trypsin. This result demonstrates that beta-fac tor-XII(a) prefers an arginine residue over lysine residue, while tryp sin is non-specific to lysine or arginine in its binding pocket. On th e other hand, the specificity of CMTI-V could be changed from trypsin to chymotrypsin inhibition by mutation of the P1 residue to either leu cine or methionine (K44L or K44M). (C) 1995 Academic Press, Inc.