Effect of P-2 ' site tryptophan and P-20 ' site deletion of Momordica charantia trypsin inhibitor II on inhibition of proteinases

Momordica charantia trypsin inhibitor II (MCTI-II) inhibits the amidolytic activity of factor Xa with a K-i value 10-100-fold smaller than those of ot her squash family inhibitors. It also inhibits factor X activation mediated by factor VIIa-tissue factor complex or factor IXa. Comparison of other sq uash family inhibitors reveal Trp at position 7 (P-2') and a deletion at po sition 25 (P-20') are characteristics of MCTI-II. In order to elucidate the effect of these positions on the inhibitory activity, wt chemically synthe sized three inhibitors: S-MCTI-II whose amino acid sequence is identical to natural MCTI-II, S-MCTI-II(7L) whose P-2'(Trp) is substituted with Leu, an d S-MCTI-II(25N) whose P-20'(deletion) is filled with Asn. The dissociation constants of the complexes of human factor Xa with S-MCTI-II, S-MCTI-II(7L ), and S-MCTI-II(25N) were 1.3 x 10(-6) M, 2.8 x 10(-5) M, and 7.3 x 10(-6) M, respectively. They inhibited factor X activation mediated by factor Wa with the same degree. As in the case of natural MCTI-II, S-MCTI-II suppress ed factor X activation mediated by factor IXa, while S-MCTI-II(7L) and S-MC TI-II(25N) did not. Both the Trp at the P-2' position and deletion at the P -20' position are thus likely required for the inhibition of factor Xa, try psin, and factor IXa, while these two positions do not affect factor X acti vation initiated by the factor VIIa-tissue factor complex. (C) 2000 Elsevie r Science B.V. All rights reserved.