Chemical modification of carboxyl groups in porcine pepsin.

Carboxyl groups i n porcine pepsin were chemically modified "by the carbodiimide reaction using waterrsoluble l-ethyl-3-(3-dimethylaminopropyl) carbodiimide and amino acid esters as nucleophiles. The modification resulted in profound changes in the a c t i v i t i e s , specificity and.some physicochemical properties of the enzyme. These include* (1) significant decrease in milk clotting activity without changes i n proteolytic activity against hemoglobin; (2) decrease i n peptidase activity against N-acetyl-L-phenylalanyl-diiodoL-tyrosine; (3) increase in clotting activity against Xcasein but decrease i n clotting activity against Kcasein mixture; (k) s h i f t -in proteolytic pH profile with pH optimum increased from 2.0 to about 3*5; (5.) decrease i n relative electrophoretic mobility and a slight decrease in isoelectric point; (6) increase i n K m without much change i n k c a i ;; and (7) increase i n s t a b i l i t y at pH above 6.0. Results suggest that the drop i n milk clotting activity was due to a change i n the charge distribution on the enzyme affecting enzyme-micelle interaction. The presence of dipeptide substrates interfered with the carboxyl modification suggestive of the proximity of the modified groups to the enzyme active s i t e .