E. Varallyay et al., 2 MUTATIONS IN RAT TRYPSIN CONFER RESISTANCE AGAINST AUTOLYSIS, Biochemical and biophysical research communications, 243(1), 1998, pp. 56-60
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.