Degradation of Escherichia coli polynucleotide phosphorylase by E. coli endogenous proteases and by trypsin

Abstract

Summary Occurrence of proteolysis of E. coli B polynucleotide phosphorylase by endogenous proteases, and degradation by isolated E. coli protease I have been demonstrated. These degradations have been compared to trypsin digestion. The proteolyzed active species have higher electrophoretic mobilities on polyacrylamide gels than the native polynucleotide phosphorylase. In every case, extensive degradation leads to the complete transformation of the enzyme into a species which migrates twice as fast as the native enzyme on 7.5 p. 100 gels. The thermal stability of the degraded enzymes decreases as the amount of degradation increases, while the molecular weight of the enzyme species also decreases (from 200 000 for the native enzyme to 165 000 for the most degraded species, as determined by the method of Hedrick and Smith ). The degradation also results in a change of catalytic properties: the primer-dependency of ADP polymerization and of ADP/32P1 exchange increases; at the same time, the ability of poly A to overcome the lag phase of ADP polymerization is lost and the KM of poly A in the phosphorolysis reaction is greatly increased. Contrarywise, the affinity for oligonucleotides is not as markedly changed (Table I). As with native enzyme, protein kinase treatment can replace the action of oligonucleotide for restoring the enzyme activity in the polymerization reaction. Thiols are without effect on the activity of the degraded enzymes. The present article underlines the interest of enzyme limited proteolysis as a tool for studying the structure-mechanism relationship. The degradation of E. coli polynucleotide phosphorylase by a protease from the same organism might serve as a model for understanding the in vivo function of proteases.