Patarroyo-Vargas Adriana M, Merino-Cabrera Yaremis B, Zanuncio Jose C, Rocha Francelina, Campos Wellington G, de Almeida Oliveira Maria Goreti
Departamento de Bioquimica e Biologia Molecular, Universidade Federal de Vicosa, 36570-000 Vicosa, Minas Gerais. Brazil.
Departamento de Entomologia/BIOAGRO, Universidade Federal de Vicosa, 36570-000 Vicosa, Minas Gerais. Brazil.
Protein Pept Lett. 2017;24(11):1040-1047. doi: 10.2174/0929866524666170918103146.
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.
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.
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.
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.
酶动力学有助于理解昆虫消化丝氨酸蛋白酶的结构和功能。动力学参数有助于了解酶的活性位点和作用机制，识别植物产生的抑制剂或通过应用合成抑制剂对昆虫消化蛋白酶系统的抑制作用。目的：本研究旨在纯化棉铃虫消化丝氨酸蛋白酶，使用生色底物三肽基测定其动力学性质，并表征合成抑制剂对其活性的影响。以便通过开发蛋白酶抑制剂为可持续害虫管理提供新机会。
在FPLC系统中，使用12.5%聚丙烯酰胺凝胶电泳，在对氨基苯甲脒琼脂糖亲和柱上纯化酶。根据狄克逊方法和林-贝氏双倒数法测定米氏常数和抑制模型。
肽底物的KM值和催化常数表明，棉铃虫类胰蛋白酶对P1位置含有精氨酸的底物具有更高的亲和力。在L-BApNA存在下，Gor 3、Gor 4和Gor 5的抑制作用呈线性竞争。Gor 5肽的抑制常数较高，因为它与棉铃虫类胰蛋白酶二级位点区域的疏水残基有强烈相互作用。
观察到在所分析的三种肽中，Gor 5的抑制常数较低，因此在测试的肽中最有效。酶二级位点区域中疏水残基的优势有利于肽的相互作用。通过三种不同类型的图谱表征后，可以验证在所分析的抑制剂和底物浓度范围内，类胰蛋白酶对测试肽的抑制模型为线性竞争型。然而，通过图谱观察到，抑制作用是由于肽在被分析酶疏水腔内的二级位点S2'处相互作用而发生的。
