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Enzymes enhance chemical reaction rates by lowering the activation energy, the energy barrier of the reaction leading to products. This occurs because enzymes bind the high-energy intermediate of the reaction (the transition state) more strongly than the substrate. We studied details of this process by determining the substrate binding energy (DeltaG(s), calculated from K(m) values) and the activation energy (DeltaG(T), determined from k(cat)/K(m) values) for the trypsin-catalyzed hydrolysis of oligopeptides. Plots of DeltaG(T) versus DeltaG(s) for oligopeptides with 15 amino acid replacements at each of the positions P(1)', P(1), and P(2) were straight lines, as predicted by a derived equation that relates DeltaG(T) and DeltaG(s). The data led to the conclusion that the trypsin active site has subsites that bind moieties of substrate and of transition state in characteristic ratios, whichever substrate is used. This was unexpected and means that each subsite characteristically favors substrate binding or catalysis.

1. 1. The properties of β-acetylglucosaminidase, acid phosphatase, aminopeptidase, cathepsin B-like proteinase, α-galactosidase, α-glucosidase, detergent-solubilized membrane-bound α-glucosidase, β-glucosidase, α-mannosidase and detergent-solubilized membrane-bound α-mannosidase from posterior midgut of Rhodnius prolixus adults were determined by density-gradient centrifugation and kinetic methods. 2. 2. The posterior midgut hydrolases from R. prolixus adults and nymphs were found to be very similar. 3. 3. The results lend support to the hypothesis that digestive enzymes are, in contrast to holometabolous insects, identical in young and adult forms of hemimetabolous insects.

Tripsinas e quimotripsinas são classes de serina peptidases amplamente estudadas e fortemente responsáveis pela digestão proteica, pela clivagem de ligações peptídicas no lado carboxila de L-aminoácidos de cadeia lateral básica e hidrofóbica, respectivamente. Três processos regulam finamente a ação dessas peptidases: secreção, ativação do precursor (zimogênio) e o sítio de reconhecimento do substrato. No presente trabalho é apresentada uma análise filogenética detalhada das tripsinas e quimotripsinas de três ordens de insetos holometábolos, revelando características divergentes nas enzimas de Lepidóptera em relação a Coleóptera e Díptera. Em particular, o sub-sítio S1 das tripsinas foi observado como mais hidrofílico em Lepidóptera do que em Coleóptera e Díptera, enquanto os sub-sítios S2-S4 parecem mais hidrofóbicos, sugerindo diferente preferências pelo substrato. Além disso, Lepidóptera mostrou um grupo de tripsinas bastante específico a um grupo taxonômico, compreendendo somente proteínas de espécies da família Noctuidae. Evidências de eventos de auto-ativação facilitada foram também observadas em todas as ordens de insetos estudadas, com as características do motivo de ativação do zimogênio complementárias ao sítio ativo das tripsinas. Em contraste, as quimotripsinas de insetos não parecem ter uma história evolutiva peculiar com respeito a, por exemplo, seus homólogos em mamíferos. Em geral, os presentes resultados sugerem que a necessidade de uma rápida taxa de autoativação fez os insetos holometábolos selecionarem grupos especializados de tripsinas com altas taxas de auto-ativação e também destacam que a evolução das tripsinas culminou em um grupo especializado de enzimas em Lepidóptera. Trypsins and chymotrypsins are well-studied classes of serine peptidases largely responsible for the digestion of proteins by cleavage of the peptide bond at the carboxyl side of basic and hydrophobic L-amino acids, respectively. Three processes mainly regulate the action of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. In the present work is presented a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects revealing divergent characteristics in the Lepidoptera enzymes in relation to Coleoptera and Diptera. In particular, trypsin subsite S1 was observed to be more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 appeared more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a very specific taxonomic trypsin group, only encompassing proteins from the Noctuidae family. Evidences for facilitated trypsin auto-activation events were also observed in all the insect orders at hand, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to e.g. their mammal counterparts. Overall, the present findings suggest that the need for fast digestion made holometabolous insects evolve specialized groups of trypsins with high autoactivation rates and highlight that the evolution of trypsins culminated in a specialized group of enzymes in Lepidoptera.

Spiders and scorpions are efficient predators arachnid (PA) consuming preys larger than themselves. Few studies reported, molecularly, the digestion in PA. This work describes a biochemical, transcriptomic and proteomic analysis of the midgut and midgut glands (MMG) and digestive juice (DJ) from Nephilengys cruentata and Tityus serrulatus MMG. Cathepsin L, B, D and F, legumain, trypsin, astacin, carbohydrases and lipases were identified by these approaches. Peptide isomerase and ctenitoxins, which are venom proteins were identified, showing a correlation among digestive and venom enzymes. Summarily, PA relies in multi peptidase system mainly constituted of astacins for extracellular prey liquefaction and cathepsin L for intracellular digestion, describing a molecular model for digestion. Probably, during evolution, gene duplication led a diversification of astacins in the derived groups of Arachnida, like spiders, distinctly from what is observed in basal groups like scorpions. These data on Arachnida digestion will allow detailed multi disciplinary studies. Aranhas e escorpiões são aracnídeos predadores eficientes (AP) consumindo presas maiores que eles mesmos. Poucos estudos descrevem molecularmente a digestão em AP. Neste trabalho caracterizamos bioquimicamente, por transcriptoma e proteoma o intestino e glândulas digestivas (IGD) e suco digestivo (SD) de Nephilengys cruentata e o IGD de Tityus serrulatus. Catepsinas L, B, D e F, legumaína, tripsinas, astacinas, carboidrases e lipases foram identificadas. Peptídeo isomerase e ctenitoxina foram identificadas no IGD. Estas proteínas podem indicar uma correlação entre enzimas digestivas e do veneno. Portanto, AP apresentam várias peptidases principalmente astacinas para liquefazer a presa extraoralmente e catepsinas L para digestão intracelular, descrevendo um modelo molecular para a digestão. Provavelmente, durante a evolução, eventos duplicação gênica levaram à diversificação das astacinas aracnídeos derivados, como as aranhas, diferentemente dos grupos basais, como os escorpiões. Estes dados sobre a digestão em Arachnida permitirão estudos multidisciplinares.