Insect chymotrypsins: chloromethyl ketone inactivation and substrate specificity relative to possible coevolutional adaptation of insects and plants
Autor: | Adriana R. Lopes, Walter R. Terra, Paloma Mieko Sato |
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Rok vydání: | 2009 |
Předmět: |
Insecta
Physiology Protein digestion Biochemistry Amino Acid Chloromethyl Ketones Substrate Specificity Affinity chromatography Animals Chymotrypsin Plant Physiological Phenomena Alanine chemistry.chemical_classification biology fungi Active site General Medicine Adaptation Physiological Amino acid Kinetics Enzyme chemistry Insect Science Glycine biology.protein Electrophoresis Polyacrylamide Gel Trypsin Inhibitors |
Zdroj: | Archives of Insect Biochemistry and Physiology. 70:188-203 |
ISSN: | 1520-6327 0739-4462 |
Popis: | Insect digestive chymotrypsins are present in a large variety of insect orders but their substrate specificity still remains unclear. Four insect chymotrypsins from 3 different insect orders (Dictyoptera, Coleoptera, and two Lepidoptera) were isolated using affinity chromatography. Enzymes presented molecular masses in the range of 20 to 31 kDa and pH optima in the range of 7.5 to 10.0. Kinetic characterization using different colorimetric and fluorescent substrates indicated that insect chymotrypsins differ from bovine chymotrypsin in their primary specificity toward small substrates (like N-benzoyl-L-Tyr p-nitroanilide) rather than on their preference for large substrates (exemplified by Succynil-Ala-Ala-Pro-Phe p-nitroanilide). Chloromethyl ketones (TPCK, N- α-tosyl-L-Phe chloromethyl ketone and Z-GGF-CK, N- carbobenzoxy-Gly-Gly-Phe-CK) inactivated all chymotrypsins tested. Inactivation rates follow apparent first-order kinetics with variable second order rates (TPCK, 42 to 130 M−1 s−1; Z-GGF-CK, 150 to 450 M−1 s−1) that may be remarkably low for S. frugiperda chymotrypsin (TPCK, 6 M−1 s−1; Z-GGF-CK, 6.1 M−1 s−1). Homology modelling and sequence alignment showed that in lepidopteran chymotrypsins, differences in the amino acid residues in the neighborhood of the catalytic His 57 may affect its pKa value. This is proposed as the cause of the decrease in His 57 reactivity toward chloromethyl ketones. Such amino acid replacement in the active site is proposed to be an adaptation to the presence of dietary ketones. © 2009 Wiley Periodicals, Inc. |
Databáze: | OpenAIRE |
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