The coevolutionary arms race of plants and insects is a complex system, including a large number of adaptations. The binding and specificity of plant proteinase inhibitors towards proteinases depend on hydrophobic interactions, and the amino acid residue present at P1 position in the inhibitor reactive site, often a lysine residue. Serine endopeptidases are the most important digestive proteinases in the majority of the insect orders. In order to determine insect trypsin specificity and to propose trypsin evolutionary pathways, we purified the digestive trypsins from Periplaneta americana (Dictyoptera), Tenebrio molitor (Coleoptera), Musca domestica (Diptera) and Diatraea saccharalis (Lepidoptera). These enzymes were kinetically characterized by using quenched fluorescent oligopeptides with 15 amino acid replacements at each of the positions P1?, P2, P3 and 2 amino acid replacements at position P1. The hydropathic index of each binding subsite (S2, S3 e S1?) was determined, based on their preference for amino acid residues. The results indicate that the degree of hydrophobicity in insect trypsin subsities increased during evolution of insect orders. Furthermore, there was an important change in S1 specificity in the Lepidopteran trypsin, which preferentially cleaves a substrate containing a Lys rather than an Arg residue at P1 position, thus differing from other insect and mammalian trypsins. The increase in hydrophobicity and the change in specificity of trypsin seem to have evolved in order to be able to transform plant protein protease inhibitors from slow hydrolysing substrates into good substrates, indicating a coevolutionary process between insect trypsins and plant protease inhibitors.
Supported by the Brazilian Research agencies FAPESP, CNPq and PRONEX.
Keywords: insect digestion
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