Activity of the reduced zymogen of streptococcal proteinase.

Abstract

Reduction of the protein—S—S—R bond in the zymogen of streptococcal proteinase leads to an enzymatically active molecule without any proteolysis being required. This conclusion is based upon experiments in which the zymogen has been reduced in very dilute solution where the rate of bimolecular autodigestion is at a minimum. Under these conditions, even in the presence of 3.5 M guanidinium chloride, the rate of activation depends upon the concentration of reducing agent and does not depend upon the protein concentration; also, the amino acid composition of the activated zymogen is indistinguishable from that of the starting material. In the more concentrated solutions that have usually been employed for autodigestion, reduced zymogen molecules can initiate the autodigestion that leads to removal of about 100 amino acid residues. The end product in that case has been shown earlier to be similar to that obtained when the conversion is accomplished by controlled proteolysis with trypsin followed by reduction of the protein—S—S—R bond.

In aqueous solution, the rate of reduction of the disulfide bond in the zymogen is slow relative to the rate of its reduction in the molecule obtained after proteolysis. The slowness of reduction contributes to the stability of the zymogen in dilute solutions under mildly reducing conditions such as may exist in bacterial cultures in the early stages of growth. The rate of reduction can be accelerated in vitro by the partial unfolding of the molecule induced by the addition of guanidinium chloride. The reduced zymogen and the reduced proteolyzed zymogen possess similar specific activities.

The nature of the zymogen-enzyme relationship for this enzyme of bacterial origin differs from that which has been established for the zymogens of pancreatic proteinases where proteolysis is a prerequisite for activity.