Chemical modification of carboxyl groups in porcine pepsin

Abstract

Modification of up to 11 carboxyl groups in porcine pepsin with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and glycine methyl ester caused changes in activities, specificity, and physicochemical properties of the enzyme. The milk clotting activity was markedly decreased to 10%, while the proteolytic activity was not affected. The decrease in the peptidase activity was about 50%. The charge density of pepsin decreased upon modification, as shown in a decrease of relative electrophoretic mobility and in a shift of pH optimum from 2 to 3.5. Kinetic studies showed that K m was increased, while k cat was not significantly affected. The presence of dipeptide substrates interfered with the modification. The modified pepsin remained reactive to two site-specific pepsin inhibitors. These effects of carboxyl modification were not unique to pepsin; modification of carboxyl groups caused similar changes in the activities and properties of pepsinogen and chymosin. The stability of the modified pepsin near neutral pH was considerably improved, suggesting that the modified enzyme may be a more suitable rennet substitute than native pepsin in cheese-making. © 1980 American Chemical Society.