Binding of the Bovine Basic Pancreatic Trypsin Inhibitor (Kunitz Inhibitor) to Human and Bovine Factor Xa. A Thermodynamic Study

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Abstract
The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human and bovine factor Xa (Stuart-Prower factor; EC 3.4.21.6) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to human and bovine factor Xa are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0.degree. C) for BPTI binding to human and bovine factor Xa decrease, thus reflecting the acidic pK shift of the His57 catalytic residue from 7.1, in the free enzyme, to 5.2, in the proteinase-inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human and bovine factor Xa are: Ka = 2.1 .times. 105 M-1 (at 21.0.degree. C), .DELTA.Go= -29.7 kJ/mol (at 21.0 .degree.C), .DELTA.So = + 161 entropy units (at 21.0 .degree. C), and .DELTA.Ho = + 17.6 kJ/mol (temperature-independent over the explored range, from 5.0.degree. C to 45.0.degree. C). Thermodynamics of BPTI binding to human and bovine factor Xa have been analysed in parallel with those of related serine (pro)enzyme/Kazal- and/Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human and bovine factor Xa was related to the inferred stereochemistry of the proteinase-inhibitor contact region.