Inhibition kinetics of digestive proteases for Anticarsia gemmatalis

Abstract

Anticarsia gemmatalis Hübner, 1818 (Lepidoptera) is a major pest of soybean in the Brazil. It is known that the reduction of proteolytic activity by the ingestion of protease inhibitors reduces digestion and larval development of the insects. Control via inhibition of the digestive enzymes necessitates deeper knowledge of the enzyme kinetics and the characterization of the inhibition kinetics of these proteases, for better understanding of the active centers and action mechanisms of this enzyme. Trypsin-like proteases found in the gut of Anticarsia gemmatalis were purifi ed in a p-aminobenzamidine agarose column. Kinetic characterization showed K M 0.503 mM for the L-BApNA substrate; V max = 46.650 nM s -1 ; V max /[E]= 9.256 nM s -1 mg L -1 and V max /[E]/K M = 18.402 nM s -1 mg L -1 mM. The K i values for the inhibitors benzamidine, berenil, SKTI and SBBI were 11.2 µM, 32.4 µM, 0.25 nM and 1.4 nM, respectively, and all revealed linear competitive inhibition. The SKTI showed the greatest inhibition, which makes it a promising subject for future research to manufacture peptide mimetic inhibitors.

Extensive reprogramming of protein isoforms and histopathological alterations in the midgut of Anticarsia gemmatalis fed with protease inhibitors

“…At the kinetic level, SKTI show lower inhibition constant (Ki) compared with those from synthetic inhibitors, which allow them to occupy all the binding sites available in the active centre of the enzyme and thus an optimised adjustment occurs in the enzyme‐inhibitor (EI) complex formation. The synthetic inhibitors, being smaller, do not occupy all the available sites of the enzyme, forming less stable EI complex (Meriño‐Cabrera, de Oliveira Mendes, et al, 2020; Meriño‐Cabrera, Severiche Castro, et al, 2020; Patarroyo‐Vargas et al, 2020; Volpicella et al, 2003). In this study, we found that protein synthesis is accompanied by changes in the insect's enzymes profile.…”

“…In fact, potency and specificity for different serine proteases can be attained by incorporating the small inhibitor benzamidine into larger and more complex synthetic inhibitors such as a bis-benzamidine, an active compound of Berenil ® . This inhibitor is an example of a synthetic trypsin inhibitor (Oliveira et al, 1993) but its potential effect as insecticide, repellent and antifeedant against pests has been poorly tested in comparison with the natural soybean inhibitor SKTI (Marinho-Prado et al, 2012;Patarroyo-Vargas et al, 2020).…”

Inhibition constant and stability of tripeptide inhibitors of gut trypsin‐like enzyme of the soybean pest Anticarsia gemmatalis

“…The inhibitory effect of the peptides (GORE1 and GORE2) was studied for 12 days at room temperature (25–30°C). All the experimental analyzes were compared with the known trypsin inhibitor Soybean Kunitz trypsin inhibitor (SKTI), was used because it is a stable protein that under physiological conditions binds to the active site of tipsins of soybean pests such as A. gemmatalis and has been characterized by being a competitive inhibitor (Koide et al, 1973; Patarroyo‐Vargas et al, 2020). Three aqueous solutions (6.5 mg/ml) of inhibitors were prepared.…”