The interaction between alpha-chymotrypsin and pancreatic trypsin inhibitor (Kunitz inhibitor). Kinetic and thermodynamic properties.

Abstract

1. The association constant, Ka, of the 1:1 complex formed between α‐chymotrypsin and the pancreatic trypsin inhibitor is 110 μM−1 at pH 8.0 and 25 °C. The second‐order rate constant for the association, ka, is 110 mM−1 s−1 and the first‐order rate constant for the dissociation, kd, is 10−3 s−1 under the same conditions. Thermodynamic parameters for complex formation at 25 °C, pH 8.0 are ΔGa0= ‐11.0 kcal×mol−1, ΔHa0= 3 kcal×mol−1 and ΔSa0= 48 cal. · mol−1· K−1.2. Temperature and pH‐dependences of the rate constants ka and kd have been studied.3. The difference in stability between the α‐chymotrypsin · inhibitor complex and the trypsin · inhibitor complex (Ka= 16 pM−1, ΔGa0=–18.1 kcal×mol−1) is due essentially to differences of kd values. The dissociation of the α‐chymotrypsin · inhibitor complex is 1.5×104 times faster than that of the trypsin · inhibitor complex. Differences in Ka and kd values are discussed in terms of differences in stabilizing interactions.4. The Cys14‐Cys38 disulfide bridge of the inhibitor, which is highly succeptible to reduction when the inhibitor is free, is masked in the α‐chymotrypsin · inhibitor complex. Reduction of the Cys14‐Cys38 bridge in the free inhibitor does not prevent association with α‐chymotrypsin. Values of ka and kd for the formation of the 1:1 α‐chymotrypsin · reduced‐inhibitor complex and for the complex formed with the native inhibitor are very similar. This situation differs from that observed with the trypsin · pancreatic‐inhibitor complex. In that case, reduction of Cys14‐Cys38 bridge considerably decreases the stability of the complex formed with trypsin; Ka is decreased by a factor of 3×104 and kd is increased by a factor of 8.6×103.