Effects of Protease Inhibitors on Protein Breakdown in Escherichia coli

Abstract

Abstract In an attempt to learn more about the proteolytic enzymes responsible for protein turnover in cells, the effects of various protease inhibitors on protein catabolism in Escherichia coli were examined. Diisopropyl fluorophosphate, phenylmethane sulfonyl fluoride, and toluenesulfonyl fluoride, all of which inhibit serine proteases, were found to decrease protein breakdown in E. coli, starved for a carbon source. These compounds partially inhibited protein breakdown at doses which did not inhibit growth on minimal medium. The effects of the sulfonyl fluorides were reversed by washing the cells. Protein breakdown in starving cells could also be inhibited reversibly by the aromatic diamidines, dibromopropamidine and pentamidine, which are potent trypsin inhibitors. The effects of pentamidine and p-toluenesulfonyl fluoride did not require concomitant protein synthesis. Tosyl lysine chloromethyl ketone, an inhibitor of trypsin, also inhibited protein breakdown in starving cells. A number of other protease inhibitors were tested and found either to have no effect on intact cells or to prevent normal cell growth. These findings suggest that at least one of the enzymes responsible for the increased protein breakdown in starving cells is a serine protease, possibly similar to trypsin. In cells deprived of a carbon or nitrogen source, synthesis of new enzymes should be dependent upon the breakdown of previously existent cell proteins. Treatment with the sulfonyl fluorides or aromatic diamidines inhibited amino acid incorporation into proteins under such conditions, but did not do so in growing cells. Similarly these compounds reversibly inhibited the induction of β-galactosidase and tryptophanase in cells deprived of a carbon or nitrogen source, but did not do so in cells growing on glycerol. The sulfonyl fluorides, the aromatic diamidines, and tosyl lysine chloromethyl ketone did not reduce the relatively low amount of protein breakdown found in growing cells. Growing cells degrade selectively or aberrant proteins, and the rate of protein breakdown in such cells increased markedly when they were grown on amino acid analogs, canavanine or threo-α-amino-β-chlorobutyric acid. This accelerated degradation also was not inhibited by the sulfonyl fluorides or the diamidines. These results thus suggest that there may exist at least two proteolytic systems in E. coli: one operating in all cells, which can degrade abnormal proteins, and one found in starving cells which is sensitive to sulfonyl fluorides and aromatic diamidines.