Inhibition kinetics of digestive proteases for Anticarsia gemmatalis.

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 purified in a p-aminobenzamidine agarose column. Kinetic characterization showed KM 0.503 mM for the L-BApNA substrate; Vmax= 46.650 nM s-1; Vmax/[E]= 9.256 nM s-1 mg L-1 and Vmax/[E]/KM= 18.402 nM s-1 mg L-1 mM. The Ki 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.

visGReMLIN: graph mining-based detection and visualization of conserved motifs at 3D protein-ligand interface at the atomic level.

BACKGROUND: Interactions between proteins and non-proteic small molecule ligands play important roles in the biological processes of living systems. Thus, the development of computational methods to support our understanding of the ligand-receptor recognition process is of fundamental importance since these methods are a major step towards ligand prediction, target identification, lead discovery, and more. This article presents visGReMLIN, a web server that couples a graph mining-based strategy to detect motifs at the protein-ligand interface with an interactive platform to visually explore and interpret these motifs in the context of protein-ligand interfaces. RESULTS: To illustrate the potential of visGReMLIN, we conducted two cases in which our strategy was compared with previous experimentally and computationally determined results. visGReMLIN allowed us to detect patterns previously documented in the literature in a totally visual manner. In addition, we found some motifs that we believe are relevant to protein-ligand interactions in the analyzed datasets. CONCLUSIONS: We aimed to build a visual analytics-oriented web server to detect and visualize common motifs at the protein-ligand interface. visGReMLIN motifs can support users in gaining insights on the key atoms/residues responsible for protein-ligand interactions in a dataset of complexes.

Kinetic Characterization of Anticarsia gemmatalis Digestive Serine- Proteases and the Inhibitory Effect of Synthetic Peptides.

BACKGROUND: Enzyme kinetics contributes to understanding the structure and function of insect digestive serine proteases. Kinetic parameters allow to understanding active sites and mechanisms of enzymes efficacy, identifying the inhibition of the insects digestive protease system by inhibitors produced by plants, or via the application of synthetic inhibitors Objectives: The aim of this study was to purify digestive serine proteases of A. gemmatalis, determining their kinetic properties using the chromogenic substrates tripeptidyl and characterizing the effects of synthetic inhibitors on their activity. In order to provide new opportunities for sustainable pest management through the development of protease inhibitors. METHODS: The enzymes were purified on p-aminobenzamidine agarose affinity column in an FPLC system using electrophoresis with 12.5% polyacrylamide gel. Michaelis-Menten constants and the inhibition model were determined according to the Dixon methodology and Lineweaver-Burk's double reciprocal. RESULTS: The KM values and catalytic constants of peptide substrates show that A. gemmatalis trypsin- like has a higher affinity for substrates with arginine in the P1 position. Inhibition by Gor 3, Gor 4, and Gor 5, in the presence of L-BApNA, was linear competitive. The inhibition constant for the Gor 5 peptide was higher due to its strong interaction with hydrophobic residues in the secondary site region of A. gemmatalis trypsin-like. CONCLUSION: It is observed that among the three peptides analyzed, the Gor 5 presented lower inhibition constant and therefore, the most potent among the tested ones. The predominance of hydrophobic residues in the region of the secondary site of the enzymes favored the interaction of the peptide. After characterization by three different types of graphs profiles, it is possible to verify that the inhibition model of the trypsin-like enzymes for the tested peptides is of the linear competitive type, in the concentration range of inhibitors and substrates analyzed. However, by the graphing profiles it is observed that the inhibition occurred due to the interaction of the peptides at the secondary site S2' in the hydrophobic cavity of the enzymes analyzed.