Thermostable and active phosphoenolpyruvate carboxylase from Thermus sp. even after proteolytic cleavage

Limited proteolysis of recombinant phosphoenolpyruvate carboxylase (PEPC) from Thermus sp. (ThPEPC) with either trypsin or chymotrypsin cleaved the enzyme into major fragments of about 70 and 20 kDa. Both proteases cleaved ThPEPC in the same chain region, which is suggested to be a flexible loop near the active site. The cleavage sites by trypsin and the site by chymotrypsin were immediately adjacent. Trypsin-treated ThPEPC remained active, whereas, the activity of chymotrypsin-treated ThPEPC was essentially not detectable. Both of the cleavages had substantially no effect on the enzyme’s quaternary structure, secondary structure, and thermostability. Therefore, the effect of the cleavage on enzyme activity was dependent on the cleavage sites which are apart from each other by only one residue, while that on the tertiary structure and thermostability of ThPEPC was not.