Autocatalytic Activation of Trypsinogen by Trypsin Salvatore F. Russo and Erin T. Wahl Western Washington University, Bellingham. WA 98225 This 3-hour experiment provides an example of the biologically important process of zymogen activation. It utilizes an active site titrant for trypsin based on the acyl enzyme mechanism for this enzyme. The activation of an inactive zymogen to a functioning enzyme is important in digestion, blood coagulation, fibrinolysis, complement activation, hormone production, metamorphosis, fertilization,,and supramolecular assembly ( I ) . The activation process provides a prompt and irreversible response in these systems. The zvmoeens secreted hv the nancreas durine dieestion have been w i l characterized: ~ h e ~ b ~ s i o l o ~a&&on ical of trvosinoeen is initiated bv entero~entidase.This enzvme is se"&etedfrom the brush border ofthe small intestine whereas trvosinogen orkinates in the acinar cells of the Dancreas. ~ h u i , . t h eGte of generation of active trypsin is r e s h . e d to the confluence of these two secretory streams. Active trypsin can then act as the catalyst in the limited hydroly& of chymotrypsinogen and procarhoxypeptidase. Trypsin is specific for the hydrolysis of peptide bonds when the carhonyl is contributed by either a lysine or an arginine residue (2). In this experiment, the activation of trypsinogen is catalyzed by added trypsin
+
H20 ~rypsinogen-
Trypsin + Hexapeptide
In an autocatalytic process, the reaction is catalyzed by one of the products. In this case, trypsin is produced in the reaction as well as initiating the process (3).Activity is monitored as a function of time using the ester p-nitrophenyl-prguanidinohenzoate (NPGB). This synthetic substrate is bound and hydrolyzed by trypsin due to its guanidino group which is also found in an arginine residue of a natural substrate. NPGB is hydrolyzed according to the acyl enzyme mechanism (4)
where E = enzyme, S = substrate (NPGB), ES = MichaelisMenten complex, ES' = acyl enzyme, PI = p-nitrophenolate, and P, = p-guanidinobenzoate. The burst of p-nitrophenolate can he monitored spectrophotometrically (5). This yields a direct measure of the stoichiometric, active enzyme concentration provided the rate constant for deacylation is much smaller than that for acylation and also that
KM(.~~....~)
