Two mutations in rat trypsin confer resistance against autolysis

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Dissolved trypsin activity decreases due to autodigestion, involving proteolytic cleavage of specific arginyl and lysyl peptide bonds.
• Bovine trypsin has three reported autolysis sites: Lys61-Ser62, Arg117-Val118, and Lys145-Ser146.
• Rat trypsin, a target for protein engineering, possesses only the Lys61-Ser62 and Arg117-Val118 autolysis sites.

Purpose of the Study:

• To enhance the stability of rat trypsin against autolysis by modifying key peptide bonds.
• To investigate the role of specific residues (Lys61 and Arg117) in trypsin autolysis.
• To explore the influence of calcium ions on trypsin autolysis and stability.

Main Methods:

• Site-directed mutagenesis was employed to replace Lys61 and Arg117 with Asn in rat trypsin.
• Kinetic parameters of wild-type and mutant enzymes (K61N, R117N, K61N/R117N) were determined.
• Autolysis rates were assessed, and N-terminal sequence determination was used to follow the autolysis process.

Main Results:

• Mutations K61N and R117N individually slowed the rate of autolysis compared to wild-type trypsin.
• The double mutant K61N/R117N exhibited significantly increased stability against autolysis.
• The double mutant also demonstrated decreased sensitivity to calcium ions (Ca2+).

Conclusions:

• The study proposes a model explaining the key roles of positions 61 and 117 in trypsin autolysis and Ca2+ influence.
• Engineered rat trypsin variants show enhanced stability, with the double mutant being particularly robust.
• These findings provide in vitro support for a proposed model of human hereditary pancreatitis.