DESIGN OF VARIANTS OF THE 2ND DOMAIN OF URINARY TRYPSIN-INHIBITOR (R-020) WITH INCREASED FACTOR-XA INHIBITORY ACTIVITY

The second domain (R-020) of human urinary trypsin inhibitor (UTI) exe rts similar inhibitory activities on trypsin, cr-chymotrypsin, leukocy te elastase, and plasmin to those of UTI itself, and additionally inhi bits coagulation factor Xa (FXa) and plasma kallikrein, on both of whi ch UTI has no inhibitory effect. In the present study, we attempted to increase this FXa-inhibitory activity by modeling the structure of R- 020-FXa complex and substituting one or two amino acids in R-020 using recombinant DNA technology. Molecular modeling of R-020 and FXa was p erformed with reference to X-ray analysis of the complex of bovine pan creatic trypsin inhibitor (BPTI) and bovine trypsin to determine the s ite of amino acid modification. The expression plasmids into which R-0 20 genes with base substitution were inserted were prepared and introd uced into Escherichia coli to express R-020 variants. The resulting va riants were purified and their enzyme inhibitory activities were measu red. The FXa-inhibitory activity was increased in four variants with s ingle amino acid substitution. With another four variants having two a mino acid substitutions involving combinations of the above single ami no acid substitutions, the FXa-inhibitory activity was further increas ed. Because the electrostatic interaction within R-020-FXa complex see med stronger in these R-020 variants, this increase in FXa-inhibitory effect was speculated to be a consequence of more potent binding betwe en the enzyme and the inhibitor.