Inhibition of six serine proteinases of the human coagulation system by mutants of bovine pancreatic trypsin inhibitor

series of 12 bovine pancreatic trypsin inhibitor variants mutated in the P-4 and P-3 positions of the canonical binding loop containing additional R15R and M52L mutations mere used to probe the role of single amino acid substitutions on binding to bovine trypsin and to the following human proteinases involved in blood clotting: plasmin, plasma kallikrein, factors X-a and XIIa, thrombin, and protein C. The mutants were expressed in Escherichia coli as fusion proteins with the LE1413 hydrophobic polypeptide and purified from inclusion bodies; these steps were followed by CNBr cleavage and oxidative refolding, The mutants inhibited the blood-clotting proteinases with association constants in the range of 10(3)-10(10) M-1. Inhibition of plasma kallikrein, factors X-a and XIIa, thrombin, and protein C could be improved by up to 2 orders of magnitude by the K15R substitution The highest increase in the association constant for P-3 mutant was measured for factor XIIa; P13S substitution increased the K-a value 58-fold, Several other substitutions at P-3 resulted in about 10-fold increase for factor X-a, thrombin, and protein C. The cumulative P-3 and P-1 effects on K-alpha values for the strongest mutant compared with the wild type bovine pancreatic trypsin inhibitor were in the range of 2,2- (plasmin) to 4,000-fold (factors XIIa and X-a). The substitutions at the P-4 site always caused negative effects (a decrease in the range from over 1.000- to 1.3-fold) on binding to all studied enzymes, including trypsin, Thermal stability studies showed a very large decrease of the denaturation temperature (about 22 degreesC) for all P-4 mutants, suggesting that substitution of the mild type Gly-12 residue leads to a change in the binding loop conformation manifesting itself in non-optimal binding to the proteinase active site.