2 MUTATIONS IN RAT TRYPSIN CONFER RESISTANCE AGAINST AUTOLYSIS

Due to autodigestion the activity of dissolved trypsin successively de creases. Autolysis leads to proteolytic cleavages of some arginyl and lysyl peptide bonds of the trypsin structure. Three important autolysi s sites have been reported for bovine trypsin: Lys61-Ser62, Arg117-Val 118 and Lys145-Ser146. Out of these three sites only the first two exi st in rat trypsin, an enzyme that has been the target of protein engin eering for more than ten years. In this work Lys61 and Arg117 mere rep laced by Asn via site directed mutagenesis to transform the correspond ing peptide bonds to trypsin resistant ones. Kinetic parameters of K61 N, R117N and the double mutant K61N/R117N are practically identical ca l with those of the wild-type enzyme. By contrast, the rate of autolys is of each singly-substituted species is substantially slower than wit h the parent trypsin. In particular, the double mutant shows dramatica lly increased stability against autolysis and decreased sensitivity to Ca2+. The process of autolysis has been followed by N-terminal sequen ce determination. We propose a model to explain why these two position s play a key role in autolysis and how Ca2+ can influence this process . In addition, our in vitro results strongly support the recently prop osed model of human hereditary pancreatitis. (C) 1998 Academic Press.