Inactivation of bovine trypsinogen and chymotrypsinogen by diisopropylphosphorofluoridate

Diisopropylphosphorofluoridate reacts with trypsinogen and chymotrypsinogen and inhibits the potential activity of both zymogens. The reactions follow pseudo first-order kinetics and proceed approximately four orders of magnitude slower than diisopropylphosphorylation of the corresponding enzymes. Correlation of initial rates of inactivation with incorporation of the reagent indicates that zymogen inactivation results from incorporation of 1 mol of organic phosphate per mol of protein. Peptides isolated from the active-site region of trypsinogen account for more than 60% of the label originally present in the [(14)C]diisopropylphosphoryl zymogen. It is concluded that loss of activation of trypsinogen is due to alkylphosphorylation of Ser(183). It is proposed that reduced reactivity of the zymogen, as compared to the enzyme, primarily reflects inefficient binding of substrates and inhibitors, and that Ser(183) of the active site exists in trypsinogen in an activated state.