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Biochemical characterization and structural analysis of trypsin from P lodia interpunctella midgut: implication of determinants in extremely alkaline pH activity profile
Author(s) -
Hemmati Seyed A.,
Sajedi Reza H.,
Moharramipour Saeid,
Taghdir Majid,
Rahmani Hossein,
Etezad Seyed M.,
Mehrabadi Mohammad
Publication year - 2017
Publication title -
physiological entomology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.693
H-Index - 57
eISSN - 1365-3032
pISSN - 0307-6962
DOI - 10.1111/phen.12196
Subject(s) - trypsin , enzyme , biochemistry , biology , midgut , affinity chromatography , active site , ion chromatography , botany , larva
In the present study, trypsin from P lodia interpunctella ( H übner) is characterized to discover sequence, biochemical and structural features. This enzyme is purified by ion exchange chromatography using fast protein liquid chromatography on proteins from fifth‐instar larvae. The enzyme is optimally active at 50 °C and pH 11.0. The kinetic parameters ( K m and V max ) of the enzyme are 5.3 ± 0.6 µ m and 31 ± 1.3 nmol min −1 mg −1 , respectively (using N α‐benzoyl‐ l ‐arginine ρ‐nitroanilide hydrochloride as substrate). The enzyme is inhibited by the addition of C u 2+ and M n 2+ , whereas it is activated by L i + at high concentrations. Moreover, the enzyme is almost completely inhibited in the presence of N α‐tosyl‐ l ‐lysine chloromethyl ketone hydrochloride and phenylmethanesulphonyl fluoride. To understand some characteristics of P . interpunctella trypsin, including active site structure and alkaline pH profile, a reliable structural model of P . interpunctella trypsin is built based on the F usarium oxisporum ( S chlecht) trypsin cystal structure ( Protein Data Bank code : 1GDU ). The secondary structure content of the purified trypsin from near‐ultraviolet circular dichroism data shows considerable similarities with that of P . interpunctella trypsin predicted structure. Analysis of p K a values of active site residues, a type of amino acid residue in the active site cleft and the surface charges of the model and T ribolium castaneum ( H erbst) trypsin structure as an insect species from different orders reveals some differences between them. These differences might effect on the microenvironment of the active site cleft and consequently shift its pH profile. The application of multiple theoretical and experimental techniques is well adapted to predict the enzyme structure with high accuracy and this could help in the design of a powerful inhibitor for trypsin with ideal properties.