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Abstract
A series of 12 bovine pancreatic trypsin inhibitor variants mutated in the P(4) and P(3) positions of the canonical binding loop containing additional K15R and M52L mutations were 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 XII(a), 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 XII(a), 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 XII(a); 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(a) 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 XII(a) 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 degrees C) for all P(4) mutants, suggesting that substitution of the wild type Gly-12 residue leads to a change in the binding loop conformation manifesting itself in non-optimal binding to the proteinase active site.
Highlights
Blood coagulation is a series of proteolytic events resulting in clot formation
Important are the processes of anticoagulation and fibrinolysis that are mediated by proteolytic enzymes
The interactions of protein C and plasmin with three P4 mutants were similar to those described for plasma kallikrein; the largest decrease of the Ka value occurred in the case of Val, and a smaller but still substantial decrease (30 – 40-fold) occurred upon mutation to Phe
Summary
Materials—Guanidinium chloride, urea, dimethyl sulfoxide (Me2SO), N,N-dimethylformamide, methanol, and acetonitrile were purchased from Merck. Plasma kallikrein and thrombin were dissolved in MilliQ water The concentrations of both enzymes were determined using respective molar absorbance coefficients ⑀280 ϭ 93,280 MϪ1 cmϪ1 and 24,240 MϪ1 cmϪ1 [36]. Protein C was dissolved in 50 mM Tris-HCl, 50 mM NaCl, 7.5 mM CaCl2, pH 7.4, and its concentration was determined from the 56,640 MϪ1 cmϪ1 molar absorbance coefficient at 280 nm [37]. The concentration of factor Xa was calculated from the Briggs-Haldane equation applying published values of catalytic parameters kcat and Km for the Tos-Gly-ProArg-pNA substrate [38]. The following substrates were used to measure the residual enzyme activities: Tos-Gly-Pro-Arg-pNA (bovine trypsin), H-D-Pro-Phe-Arg-pNA (␣-plasmin), Tos-Gly-Pro-Arg-pNA (factor Xa), H-D-Pro-Phe-Arg-pNA (␣-factor XIIa), Bz-Pro-Phe-Arg-pNA (plasma kallikrein), Suc-Ala-Ala-Pro-Arg-pNA (␣-thrombin), and TosGly-Pro-Arg-pNA (protein C). In the case of weak associations (Ka Ͻ 107 MϪ1) only the 2-parameter algorithm (E) ϭ f(E0, Ka) and the default value of F ϭ 1 were applied
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