The Interaction between α‐Chymotrypsin and Pancreatic Trypsin Inhibitor (Kunitz Inhibitor)

The association constant, K a , 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, k a , is 110 mM −1 s −1 and the first‐order rate constant for the dissociation, k d , is 10 −3 s −1 under the same conditions. Thermodynamic parameters for complex formation at 25 °C, pH 8.0 are Δ G a 0 = ‐11.0 kcal×mol −1 , Δ H a 0 = 3 kcal×mol −1 and Δ S a 0 = 48 cal. · mol −1 · K −1 . 2. Temperature and pH‐dependences of the rate constants k a and k d have been studied. 3. The difference in stability between the α‐chymotrypsin · inhibitor complex and the trypsin · inhibitor complex ( K a = 16 pM −1 , Δ G a 0 =–18.1 kcal×mol −1 ) is due essentially to differences of k d values. The dissociation of the α‐chymotrypsin · inhibitor complex is 1.5×10 4 times faster than that of the trypsin · inhibitor complex. Differences in K a and k d values are discussed in terms of differences in stabilizing interactions. 4. The Cys 14 ‐Cys 38 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 Cys 14 ‐Cys 38 bridge in the free inhibitor does not prevent association with α‐chymotrypsin. Values of k a and k d 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 Cys 14 ‐Cys 38 bridge considerably decreases the stability of the complex formed with trypsin; K a is decreased by a factor of 3×10 4 and k d is increased by a factor of 8.6×10 3 .