Substrate specificity of insect trypsins and the role of their subsites in catalysis

Lopes, Adriana Rios; Juliano, María A.; Marana, Sandro R.; Juliano, Luiz; Terra, Walter R.

doi:10.1016/j.ibmb.2005.11.006

Cited by 57 publications

(52 citation statements)

References 42 publications

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“…Tryptic activity was tenfold less than that found for a-chymotrypsin-like activity and even when downregulated, a-chymotryptic activity was still seven to eight times higher than tryptic activities (figure 1). Both trypsin and a-chymotrypsin occur as multiple isoforms in L. migratoria and other insects (Lam et al 1999(Lam et al , 2000Lopes et al 2006;Sato et al 2008). While the use of substrates with poor affinity for the predominant isoforms has been responsible for underestimating levels of enzyme activity, especially that of chymotrypsin (Baumann 1990;Johnston et al 1991Johnston et al , 1995Christeller et al 1992;Peterson et al 1995), the substrates we used in this study have previously been shown to be suitable for trypsin-like and a-chymotrypsinlike activity from L. migratoria GITs (Lam et al 1999(Lam et al , 2000.…”

Section: Discussionmentioning

confidence: 98%

The gastrointestinal tract as a nutrient-balancing organ

et al. 2010

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Failure to provision tissues with an appropriate balance of nutrients engenders fitness costs. Maintaining nutrient balance can be achieved by adjusting the selection and consumption of foods, but this may not be possible when the nutritional environment is limiting. Under such circumstances, rebalancing of an imbalanced nutrient intake requires post-ingestive mechanisms. The first stage at which such postingestive rebalancing might occur is within the gastrointestinal tract (GIT), by differential release of digestive enzymes-releasing less of those enzymes for nutrients present in excess while maintaining or boosting levels of enzymes for nutrients in deficit. Here, we use an insect herbivore, the locust, to show for the first time that such compensatory responses occur within the GIT. Furthermore, we show that differential release of proteases and carbohydrases in response to nutritional state translate into differential extraction of macronutrients from host plants. The prevailing view is that physiological and structural plasticity in the GIT serves to maximize the rate of nutrient gain in relation to costs of maintaining the GIT; our findings show that GIT plasticity is integral to the maintenance of nutrient balance.

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Section: Discussionmentioning

confidence: 98%

The gastrointestinal tract as a nutrient-balancing organ

et al. 2010

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“…Besides that, a systematic study of subsite specificities in insect chymotrypsin, as already done with trypsin (Lopes et al, 2006), showed differences among insect species (Sato et al, 2008) and may help in evaluating the chymotrypsin role in plant inhibitor resistance and in understanding the evolution of insect chymotrypsins.…”

Section: Chymotrypsin Ketone Inactivation and Dietary Adaptationmentioning

confidence: 84%

Insect chymotrypsins: chloromethyl ketone inactivation and substrate specificity relative to possible coevolutional adaptation of insects and plants

Lopes

Sato

Terra

2009

Arch Insect Biochem Physiol

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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- alpha-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.

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“…Sabe-se que baratas coletadas em ambiente urbano possuem pelo menos 11 diferentes atividades de serina proteinases presentes em seu intestino, entre tripsinas e quimotripsinas (Hivrale et al, 2005), e a atividade tríptica majoritária de machos cultivados em laboratório foi previamente purificada e extensivamente caracterizada bioquimicamente (Lopes et al, 2003;Marana et al, 2002;Lopes et al, 2006 (Dias, 2004 Jiang et al (1996) descreveram uma quimotripsina de Aedes aegipty envolvida com a digestão de proteínas de fêmeas adultas após alimentação. Essa quimotripsina foi expressa de maneira recombinante (sem atividade) e utilizada para produzir anticorpos policlonais em coelhos, os quais foram utilizados para analisar a presença desta proteína antes e após a alimentação.…”

Section: Serina Proteinases De Insetos E Resistência àS Defesas De Plunclassified

“…A tripsina digestiva foi purificada utilizando uma coluna de afinidade benzamidine-sepharose (Lopes et al, 2006). Após a aplicação do homogeneizado à coluna, esta foi lavada com Tris-HCl 20 mM pH 8,5 contendo NaCl 200 mM, e a atividade foi eluída com 0.1 M de benzamidina.…”

Section: Purificação De Serina Proteinasesunclassified

“…A purificação das serina proteinases de insetos da ordem Lepidoptera é usualmente realizada através da utilização de colunas de afinidade (Peterson et al, 1994;Peterson et al, 1995;Lopes et al, 2006;Valaitis, 1993). Entretanto, as atividades de serina proteinases de insetos da ordem Lepidoptera são difíceis de serem isoladas, pois geralmente há atividade contaminante de tripsina nas purificações de quimotripsinas, sendo o contrário verificado também (Peterson et al, 1994, Peterson et al, 1995.…”

Substrate specificity of insect trypsins and the role of their subsites in catalysis

Cited by 57 publications

References 42 publications

The gastrointestinal tract as a nutrient-balancing organ

The gastrointestinal tract as a nutrient-balancing organ

Insect chymotrypsins: chloromethyl ketone inactivation and substrate specificity relative to possible coevolutional adaptation of insects and plants

Serina proteinases digestivas de insetos-modelo

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