Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human Lys 77-plasmin

Abstract

The effect of pH and temperature on the association equilibrium constant ( K a) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human Lys 77-plasmin has been investigated. K a values decrease with decreasing pH, reflecting the acid-pK and -midpoint shifts, upon BPTI binding, of a single ionizable group, between pH 5 and 9, and of a three-proton transition, between pH 3 and 5. At pH 8.0, values of thermodynamic parameters for BPTI binding to human Lys 77-plasmin are: K a = 1.2×10 9 m −1, ΔG ° = − 12.2 kcal/mol, and ΔS ° = + 49 entropy units (at 21 °C); and ΔH ° = + 2.3 kcal/mol (temperature independent between 5 °C and 45 °C; l kcal = 4184 J). BPTI binding properties of human Lys 77-plasmin have been analysed in parallel with those of serine (pro)enzymes acting on cationic and non-cationic substrates. Considering the known molecular structures of homologous serine (pro)enzymes, or Kunitz and Kazal-type inhibitors and of their complexes, the observed binding behaviour of BPTI to human Lys 77-plasmin was related to the inferred stereochemistry of the enzyme-inhibitor contact region.