Comparative kinetic characterization of the activity of glycosylated and non-glycosylated trypsin-like serine protease isolated from adults of Rhyzopertha dominica (Coleoptera: Bostrichidae) reared on the grain of three different cultivars of wheat

Abstract

Rhyzopertha dominica is a pest that uses trypsin-like serine protease enzymes to hydrolyse the proteins in the cereal grains on which it feeds. The present study reveals for the fi rst time that that there are both glycosylated and non-glycosylated serine proteases. The progeny of R. dominica reared on the grain three varieties of wheat were used to fractionate their trypsinlike serine proteases using Concanavalin A affi nity chromatography. The albumin fractions from the wheat cultivars used in this study were subjected to size exclusion chromatography to fractionate the albumin inhibitors that are highly specifi c for the serine protease activity of R. dominica. Kinetic and thermodynamic assays were used to diff erentiate both types of enzymes. In general, the catalytic effi ciency values Vmax/Km for glycosylated proteases were higher, indicating that glycosylation increases the affi nity for the substrate. Inhibition assays using wheat albumins revealed that the glycosylated enzymes had higher Ki values, indicating a low affi nity for the inhibitors than the non-glycosylated enzymes. Thermodynamic analysis indicates that glycosylation increases the activation energy Ea improving the serine proteases’ catalysis. Thus it is likely that R. dominica uses glycosylated proteases in order to optimize the hydrolysis of cereal proteins and nullify the action of wheat grain protease inhibitors and increase its chances of survival. * Corresponding authors; e-mails: aldo.arvizu@unison.mx, javier.cinco@unison.mx INTRODUCTION Insects obtain essential amino acids from their food by effi cient digestive hydrolysis of plant proteins (Gholamzadeh et al., 2013; Zhu-Salzman & Zeng, 2015). Their digestive proteases catalyse the hydrolysis of proteins important for growth and development. The proteases are classifi ed according to the chemical nature of the groups responsible for catalysis, i.e., serine, threonine, cysteine, aspartic or metalloproteases (López-Otín & Bond, 2008; Barrett et al., 2012). In the digestive tract of some species of insects such Eur. J. Entomol. 120: 233–243, 2023 doi: 10.14411/eje.2023.026