Substrate specificity, physicochemical and kinetic properties of a trypsin from the giant Amazonian fish pirarucu (Arapaima gigas)

Specificity studies can contribute to the understanding of how substrate binding and catalysis change among similar enzymes. This study evaluated through the substrate specificity, physicochemical and kinetic properties of a trypsin from the pyloric caeca of pirarucu (Arapaima gigas). For such purposes, two series of FRET-peptides (Abz-RXFK-Eddnp and Abz-XRFK-Eddnp) were used. The mature trypsin molecular mass was 23.5 kDa and N-terminal sequence was IVGGYECPRNSVPYQVSLNVGYH. Moreover, higher trypsin catalytic efficiency was observed for the substrates containing Arg, Tyr and Ser at P1′ and Lys and Val at P2. However, catalytic deficiency was found in the presence of Pro, Trp, Asp, Gly, Glu, Ala at P1’ and Gly, Glu, Trp, Asn, Gln and Tyr at P2. Using z-FR-MCA as a substrate, the optimum pH (9.25) and temperature (45 °C) were determined by pseudo-first-order kinetics. The enzyme retained all of its initial activity after 180 min incubation at temperatures up to 45 °C. Kinetic assays in the presence of Ca2+ showed an increase of approximately 7-fold in enzyme catalytic efficiency (kcat/Km). On the other hand, Na+, K+ and Mg2+ were able to inhibit pirarucu trypsin. Therefore, the knowledge of kinetic and biochemical properties, as well as enzyme specificity, are important for the evaluation of biotechnological use of this enzyme and may contribute to the development of more efficient substrates for fish trypsin. [Display omitted] •N-terminal sequence of Arapaima gigas trypsin is homologous to other fish trypsins.•Pirarucu trypsin was able to hydrolyze substrates with Arg, Tyr and Ser at P1'.•Pirarucu trypsin was stable at 45 °C for 180 min using z-FR-MCA.•Calcium activated trypsin, whereas sodium, potassium and magnesium inhibited it.