Enzymatic resynthesis of the "reactive site" bond in the modified aprotinin derivatives [seco-15/16]aprotinin and [Di-seco-15/16,39/40]aprotinin

On incubation of [di-seco-15/16,39/40]aprotinin with human plasmin, porcine pancreatic kallikrein or bovine or porcine trypsin in neutral or slightly alkaline solutions [seco-39/40]aprotinin is slowly formed with enzymatic resynthesis of the reactive-site bond 15/16. With chymotrypsin, however, further degradation of [di-seco-15/16,39/40]aprotinin takes place without enzymatic resynthesis. The apparent rate constants for the synthesis of [seco-39/40]aprotinin with kallikrein and trypsin have been determined and indicate that the bond-forming reaction is 10-200-fold slower with [di-seco-15/16,39/40]aprotinin than with [seco-15/16]aprotinin. The newly formed [seco-39/40]aprotinin has similar kinetic constants for the complexation with its cognate enzymes as aprotinin, indicating that any distortion of the secondary binding region due to cleavage of the Arg39-Ala40 bond does not seriously influence binding and affinities.