Initial digestion of proteins is carried out by proteinases (endopeptidases), which are enzymes able to cleave the internal peptide bonds of proteins (Fig. 1A). Different endopeptidases are necessary to do this because the amino acid residues vary along the peptide chain (R is a variable group in Fig. 1A). Proteinases may differ in specificity toward the reactant protein (substrate) and are grouped according to their reaction mechanism into the subclasses: serine, cysteine, and aspartic proteinases. Trypsin, chymotrypsin, and elastase are serine proteinases that are widely distributed in insects and have molecular masses in the range 20 to 35 kDa and alkaline pH optima. Trypsin preferentially hydrolyzes (its primary specificity) peptide bonds in the carboxyl end of amino acids with basic R groups (Arg, Lys); chymotrypsin is preferential toward large hydrophobic R groups (e.g., Phe, Tyr) and elastase, toward small hydrophobic R groups (e.g., Ala). The activity of the enzymes also depends on the amino acid residues neighboring the bond to be cleaved. This may explain the differences in susceptibilities of insects to strains of Bacillus thuringiensis, because the deleterious effects depend on the previous proteolysis of the bacterial endotoxin. Related to this is the growing evidence that insects fed on trypsin inhibitor-containing food express new trypsin molecules insensitive to the inhibitors. These inhibitors are proteins and their binding to the enzyme has molecular requirements similar to those of the substrate.
Cysteine and aspartic proteinases are the only midgut proteinases in hemipterans and they occur in addition to serine proteinases in cucujiformia beetles. Their occurrence in Hemiptera is interpreted as a consequence of the loss of the usual digestive serine proteinases associated with the adaptation of hemipteran ancestors to a diet lacking proteins (plant sap), followed by the use of lysosome-like enzymes in adapting to a new predatory habit. The presence of cysteine and aspartic proteinases in cucujiformia beetles is likely an ancestral adaptation to circumvent proteolytic inhibition caused by trypsin inhibitors in ingested seeds. Cysteine and aspartic proteinases have pH optima of 5.5 to 6.0 and 3.2 to 3.5 and molecular masses of 20 to 40 kDa and 60 to 80 kDa, respectively. Because of their pH optima, aspartic proteinases are not very active in the mildly acidic midguts of Hemiptera and cucujiformia beetles, but are very important in the middle midguts (pH 3.5) of cyclorrhaphous flies.
Intermediate digestion of proteins is accomplished by exopeptidases, enzymes that remove amino acids from the N-terminal (aminopeptidases) or C-terminal (carboxypeptidases) ends of oligopeptides (fragments of proteins) (Fig. 1A). Insect aminopeptidases have molecular masses in the range 90 to 130 kDa, have pH optima of 7.2 to 9.0, have no marked specificity toward the N-terminal amino acid, and are usually associated with the microvillar membranes of midgut cells. Therefore, the action of aminopeptidase is restricted to the surface of midgut cells. Because aminopeptidases are frequently active on dipeptides, they are also involved in proteinterminal digestion together with dipeptidases. Aminopeptidases may account for as much as 55% of the midgut microvillar proteins in larvae of the yellow mealworm, Tenebrio molitor. Probably because of this, in many insects aminopeptidases are the preferred targets of B. thuringiensis endotoxins. These toxins, after binding to aminopeptidase (or other receptors), form channels through which cell contents leak, leading to insect death. The most important insect carboxypeptidases have alkaline pH optima, have molecular masses in the range 20 to 50 kDa, and require a divalent metal for activity. They are classified as carboxypeptidase A or B depending on their activity upon neutral/acid or basic C-terminal amino acids, respectively.
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