Limited proteolysis of porcine pancreatic and barley isozyme 2 α-amylases occurs in specific loops of their (β/α) 8-barrel domain

Abstract

α-Amylases are, in general, highly resistant to proteases. In an attempt to reveal domain/function relationships of porcine pancreatic (PPA) and barley isozyme 2 (BA-2) α-amylases, prolonged treatment with moderate to high concentrations of subtilisin and proteinase K, respectively, have been performed. From both PPA and BA-2, two larger fragments were obtained. SDS-PAGE indicated the cleavage products of PPA to be of apparent mol. wts 41 kd and 14 kd, while BA-2 gave rise to 33 kd and 12 kd fragments. The susceptible peptide bonds were identified, by sequencing of isolated fragments, to be after E 369 and Q 294 in PPA and BA-2 respectively. The X-ray structure of PPA and the predicted structure of BA-2 indicate three-domain (β/α) 8-barrel proteins. Surprisingly, the protease susceptible areas are located to different loops, namely, those following the eighth β-strand and the seventh β-strand in the (β/α) 8-barrel domains of PPA and BA-2 respectively. The limited proteolysis was accompanied by loss of enzymatic activity of both PPA and BA-2. The importance of a surface site in amylolytic activity is suggested.